Antibodies to tumor associated proteins

ABSTRACT

A novel gene 024P4C12 (also designated 24P4C12) and its encoded protein, and variants thereof, are described wherein 24P4C12 exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, 24P4C12 provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 24P4C12 gene or fragment thereof, or its encoded protein, or variants thereof, or a fragment thereof, can be used to elicit a humoral or cellular immune response; antibodies or T cells reactive with 24P4C12 can be used in active or passive immunization.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/306,631, filed 27 Nov. 2002, which is a continuation-in-part of U.S.patent application Ser. No. 09/547,789, filed 12 Apr. 2000, now U.S.Pat. No. 6,943,235, issued 13 Sep. 2005, and claims priority to U.S.provisional patent application No. 60/128,858, filed 12 Apr. 1999.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

FIELD OF THE INVENTION

The invention described herein relates to a gene and its encodedprotein, termed 24P4C12, expressed in certain cancers, and to diagnosticand therapeutic methods and compositions useful in the management ofcancers that express 24P4C12.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, as reported by the American Cancer Society,cancer causes the death of well over a half-million people annually,with over 1.2 million new cases diagnosed per year. While deaths fromheart disease have been declining significantly, those resulting fromcancer generally are on the rise. In the early part of the next century,cancer is predicted to become the leading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Furthermore, many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most commoncancer in males and is the second leading cause of cancer death in men.In the United States alone, well over 30,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, surgical castration and chemotherapy continue to be the maintreatment modalities. Unfortunately, these treatments are ineffectivefor many and are often associated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that canaccurately detect early-stage, localized tumors remains a significantlimitation in the diagnosis and management of this disease. Although theserum prostate specific antigen (PSA) assay has been a very useful tool,however its specificity and general utility is widely regarded aslacking in several important respects.

Progress in identifying additional specific markers for prostate cancerhas been improved by the generation of prostate cancer xenografts thatcan recapitulate different stages of the disease in mice. The LAPC (LosAngeles Prostate Cancer) xenografts are prostate cancer xenografts thathave survived passage in severe combined immune deficient (SCID) miceand have exhibited the capacity to mimic the transition from androgendependence to androgen independence (Klein et al., 1997, Nat. Med.3:402). More recently identified prostate cancer markers include PCTA-1(Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252),prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res1996 September 2 (9): 1445-51), STEAP (Hubert, et al., Proc Natl AcadSci USA. 1999 Dec. 7; 96(25): 14523-8) and prostate stem cell antigen(PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).

While previously identified markers such as PSA, PSM, PCTA and PSCA havefacilitated efforts to diagnose and treat prostate cancer, there is needfor the identification of additional markers and therapeutic targets forprostate and related cancers in order to further improve diagnosis andtherapy.

Renal cell carcinoma (RCC) accounts for approximately 3 percent of adultmalignancies. Once adenomas reach a diameter of 2 to 3 cm, malignantpotential exists. In the adult, the two principal malignant renal tumorsare renal cell adenocarcinoma and transitional cell carcinoma of therenal pelvis or ureter. The incidence of renal cell adenocarcinoma isestimated at more than 29,000 cases in the United States, and more than11,600 patients died of this disease in 1998. Transitional cellcarcinoma is less frequent, with an incidence of approximately 500 casesper year in the United States.

Surgery has been the primary therapy for renal cell adenocarcinoma formany decades. Until recently, metastatic disease has been refractory toany systemic therapy. With recent developments in systemic therapies,particularly immunotherapies, metastatic renal cell carcinoma may beapproached aggressively in appropriate patients with a possibility ofdurable responses. Nevertheless, there is a remaining need for effectivetherapies for these patients.

Of all new cases of cancer in the United States, bladder cancerrepresents approximately 5 percent in men (fifth most common neoplasm)and 3 percent in women (eighth most common neoplasm). The incidence isincreasing slowly, concurrent with an increasing older population. In1998, there was an estimated 54,500 cases, including 39,500 in men and15,000 in women. The age-adjusted incidence in the United States is 32per 100,000 for men and eight per 100,000 in women. The historicmale/female ratio of 3:1 may be decreasing related to smoking patternsin women. There were an estimated 11,000 deaths from bladder cancer in1998 (7,800 in men and 3,900 in women). Bladder cancer incidence andmortality strongly increase with age and will be an increasing problemas the population becomes more elderly.

Most bladder cancers recur in the bladder. Bladder cancer is managedwith a combination of transurethral resection of the bladder (TUR) andintravesical chemotherapy or immunotherapy. The multifocal and recurrentnature of bladder cancer points out the limitations of TUR. Mostmuscle-invasive cancers are not cured by TUR alone. Radical cystectomyand urinary diversion is the most effective means to eliminate thecancer but carry an undeniable impact on urinary and sexual function.There continues to be a significant need for treatment modalities thatare beneficial for bladder cancer patients.

An estimated 130,200 cases of colorectal cancer occurred in 2000 in theUnited States, including 93,800 cases of colon cancer and 36,400 ofrectal cancer. Colorectal cancers are the third most common cancers inmen and women. Incidence rates declined significantly during 1992-1996(−2.1% per year). Research suggests that these declines have been due toincreased screening and polyp removal, preventing progression of polypsto invasive cancers. There were an estimated 56,300 deaths (47,700 fromcolon cancer, 8,600 from rectal cancer) in 2000, accounting for about11% of all U.S. cancer deaths.

At present, surgery is the most common form of therapy for colorectalcancer, and for cancers that have not spread, it is frequently curative.Chemotherapy, or chemotherapy plus radiation, is given before or aftersurgery to most patients whose cancer has deeply perforated the bowelwall or has spread to the lymph nodes. A permanent colostomy (creationof an abdominal opening for elimination of body wastes) is occasionallyneeded for colon cancer and is infrequently required for rectal cancer.There continues to be a need for effective diagnostic and treatmentmodalities for colorectal cancer.

There were an estimated 164,100 new cases of lung and bronchial cancerin 2000, accounting for 14% of all U.S. cancer diagnoses. The incidencerate of lung and bronchial cancer is declining significantly in men,from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s,the rate of increase among women began to slow. In 1996, the incidencerate in women was 42.3 per 100,000.

Lung and bronchial cancer caused an estimated 156,900 deaths in 2000,accounting for 28% of all cancer deaths. During 1992-1996, mortalityfrom lung cancer declined significantly among men (−1.7% per year) whilerates for women were still significantly increasing (0.9% per year).Since 1987, more women have died each year of lung cancer than breastcancer, which, for over 40 years, was the major cause of cancer death inwomen. Decreasing lung cancer incidence and mortality rates most likelyresulted from decreased smoking rates over the previous 30 years;however, decreasing smoking patterns among women lag behind those ofmen. Of concern, although the declines in adult tobacco use have slowed,tobacco use in youth is increasing again.

Treatment options for lung and bronchial cancer are determined by thetype and stage of the cancer and include surgery, radiation therapy, andchemotherapy. For many localized cancers, surgery is usually thetreatment of choice. Because the disease has usually spread by the timeit is discovered, radiation therapy and chemotherapy are often needed incombination with surgery. Chemotherapy alone or combined with radiationis the treatment of choice for small cell lung cancer; on this regimen,a large percentage of patients experience remission, which in some casesis long lasting. There is however, an ongoing need for effectivetreatment and diagnostic approaches for lung and bronchial cancers.

An estimated 182,800 new invasive cases of breast cancer were expectedto occur among women in the United States during 2000. Additionally,about 1,400 new cases of breast cancer were expected to be diagnosed inmen in 2000. After increasing about 4% per year in the 1980s, breastcancer incidence rates in women have leveled off in the 1990s to about110.6 cases per 100,000.

In the U.S. alone, there were an estimated 41,200 deaths (40,800 women,400 men) in 2000 due to breast cancer. Breast cancer ranks second amongcancer deaths in women. According to the most recent data, mortalityrates declined significantly during 1992-1996 with the largest decreasesin younger women, both white and black. These decreases were probablythe result of earlier detection and improved treatment.

Taking into account the medical circumstances and the patientspreferences, treatment of breast cancer may involve lumpectomy (localremoval of the tumor) and removal of the lymph nodes under the arm;mastectomy (surgical removal of the breast) and removal of the lymphnodes under the arm; radiation therapy; chemotherapy; or hormonetherapy. Often, two or more methods are used in combination. Numerousstudies have shown that, for early stage disease, long-term survivalrates after lumpectomy plus radiotherapy are similar to survival ratesafter modified radical mastectomy. Significant advances inreconstruction techniques provide several options for breastreconstruction after mastectomy. Recently, such reconstruction has beendone at the same time as the mastectomy.

Local excision of ductal carcinoma in situ (DCIS) with adequate amountsof surrounding normal breast tissue may prevent the local recurrence ofthe DCIS. Radiation to the breast and/or tamoxifen may reduce the chanceof DCIS occurring in the remaining breast tissue. This is importantbecause DCIS, if left untreated, may develop into invasive breastcancer. Nevertheless, there are serious side effects or sequalae tothese treatments. There is, therefore, a need for efficacious breastcancer treatments.

There were an estimated 23,100 new cases of ovarian cancer in the UnitedStates in 2000. It accounts for 4% of all cancers among women and rankssecond among gynecologic cancers. During 1992-1996, ovarian cancerincidence rates were significantly declining. Consequent to ovariancancer, there were an estimated 14,000 deaths in 2000. Ovarian cancercauses more deaths than any other cancer of the female reproductivesystem.

Surgery, radiation therapy, and chemotherapy are treatment options forovarian cancer. Surgery usually includes the removal of one or bothovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus(hysterectomy). In some very early tumors, only the involved ovary willbe removed, especially in young women who wish to have children. Inadvanced disease, an attempt is made to remove all intra-abdominaldisease to enhance the effect of chemotherapy. There continues to be animportant need for effective treatment options for ovarian cancer.

There were an estimated 28,300 new cases of pancreatic cancer in theUnited States in 2000. Over the past 20 years, rates of pancreaticcancer have declined in men. Rates among women have remainedapproximately constant but may be beginning to decline. Pancreaticcancer caused an estimated 28,200 deaths in 2000 in the United States.Over the past 20 years, there has been a slight but significant decreasein mortality rates among men (about −0.9% per year) while rates haveincreased slightly among women.

Surgery, radiation therapy, and chemotherapy are treatment options forpancreatic cancer. These treatment options can extend survival and/orrelieve symptoms in many patients but are not likely to produce a curefor most. There is a significant need for additional therapeutic anddiagnostic options for pancreatic cancer.

SUMMARY OF THE INVENTION

The present invention relates to a gene, designated 24P4C12, that hasnow been found to be over-expressed in the cancer(s) listed in Table I.Northern blot expression analysis of 24P4C12 gene expression in normaltissues shows a restricted expression pattern in adult tissues. Thenucleotide (FIG. 2) and amino acid (FIG. 2, and FIG. 3) sequences of24P4C12 are provided. The tissue-related profile of 24P4C12 in normaladult tissues, combined with the over-expression observed in the tissueslisted in Table I, shows that 24P4C12 is aberrantly over-expressed in atleast some cancers, and thus serves as a useful diagnostic,prophylactic, prognostic, and/or therapeutic target for cancers of thetissue(s) such as those listed in Table I.

The invention provides polynucleotides corresponding or complementary toall or part of the 24P4C12 genes, mRNAs, and/or coding sequences,preferably in isolated form, including polynucleotides encoding24P4C12-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80,85, 90, 95, 100 or more than 100 contiguous amino acids of a24P4C12-related protein, as well as the peptides/proteins themselves;DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides oroligonucleotides complementary or having at least a 90% homology to the24P4C12 genes or mRNA sequences or parts thereof, and polynucleotides oroligonucleotides that hybridize to the 24P4C12 genes, mRNAs, or to24P4C12-encoding polynucleotides. Also provided are means for isolatingcDNAs and the genes encoding 24P4C12. Recombinant DNA moleculescontaining 24P4C12 polynucleotides, cells transformed or transduced withsuch molecules, and host-vector systems for the expression of 24P4C12gene products are also provided. The invention further providesantibodies that bind to 24P4C12 proteins and polypeptide fragmentsthereof, including polyclonal and monoclonal antibodies, murine andother mammalian antibodies, chimeric antibodies, humanized and fullyhuman antibodies, and antibodies labeled with a detectable marker ortherapeutic agent. In certain embodiments, there is a proviso that theentire nucleic acid sequence of FIG. 2 is not encoded and/or the entireamino acid sequence of FIG. 2 is not prepared. In certain embodiments,the entire nucleic acid sequence of FIG. 2 is encoded and/or the entireamino acid sequence of FIG. 2 is prepared, either of which are inrespective human unit dose forms.

The invention further provides methods for detecting the presence andstatus of 24P4C12 polynucleotides and proteins in various biologicalsamples, as well as methods for identifying cells that express 24P4C12.A typical embodiment of this invention provides methods for monitoring24P4C12 gene products in a tissue or hematology sample having orsuspected of having some form of growth dysregulation such as cancer.

The invention further provides various immunogenic or therapeuticcompositions and strategies for treating cancers that express 24P4C12such as cancers of tissues listed in Table I, including therapies aimedat inhibiting the transcription, translation, processing or function of24P4C12 as well as cancer vaccines. In one aspect, the inventionprovides compositions, and methods comprising them, for treating acancer that expresses 24P4C12 in a human subject wherein the compositioncomprises a carrier suitable for human use and a human unit dose of oneor more than one agent that inhibits the production or function of24P4C12. Preferably, the carrier is a uniquely human carrier. In anotheraspect of the invention, the agent is a moiety that is immunoreactivewith 24P4C12 protein. Non-limiting examples of such moieties include,but are not limited to, antibodies (such as single chain, monoclonal,polyclonal, humanized, chimeric, or human antibodies), functionalequivalents thereof (whether naturally occurring or synthetic), andcombinations thereof. The antibodies can be conjugated to a diagnosticor therapeutic moiety. In another aspect, the agent is a small moleculeas defined herein.

In another aspect, the agent comprises one or more than one peptidewhich comprises a cytotoxic T lymphocyte (CTL) epitope that binds an HLAclass I molecule in a human to elicit a CTL response to 24P4C12 and/orone or more than one peptide which comprises a helper T lymphocyte (HTL)epitope which binds an HLA class II molecule in a human to elicit an HTLresponse. The peptides of the invention may be on the same or on one ormore separate polypeptide molecules. In a further aspect of theinvention, the agent comprises one or more than one nucleic acidmolecule that expresses one or more than one of the CTL or HTL responsestimulating peptides as described above. In yet another aspect of theinvention, the one or more than one nucleic acid molecule may express amoiety that is immunologically reactive with 24P4C12 as described above.The one or more than one nucleic acid molecule may also be, or encodes,a molecule that inhibits production of 24P4C12. Non-limiting examples ofsuch molecules include, but are not limited to, those complementary to anucleotide sequence essential for production of 24P4C12 (e.g. antisensesequences or molecules that form a triple helix with a nucleotide doublehelix essential for 24P4C12 production) or a ribozyme effective to lyse24P4C12 mRNA.

Note that to determine the starting position of any peptide set forth inTables VIII-XXI and XXII to XLIX (collectively HLA Peptide Tables)respective to its parental protein, e.g., variant 1, variant 2, etc.,reference is made to three factors: the particular variant, the lengthof the peptide in an HLA Peptide Table, and the Search Peptides in TableVII. Generally, a unique Search Peptide is used to obtain HLA peptidesof a particular for a particular variant. The position of each SearchPeptide relative to its respective parent molecule is listed in TableVII. Accordingly, if a Search Peptide begins at position “X”, one mustadd the value “X−1” to each position in Tables VIII-XXI and XXII to XLIXto obtain the actual position of the HLA peptides in their parentalmolecule. For example, if a particular Search Peptide begins at position150 of its parental molecule, one must add 150-1, i.e., 149 to each HLApeptide amino acid position to calculate the position of that amino acidin the parent molecule.

One embodiment of the invention comprises an HLA peptide, that occurs atleast twice in Tables VIII-XXI and XXII to XLIX collectively, or anoligonucleotide that encodes the HLA peptide. Another embodiment of theinvention comprises an HLA peptide that occurs at least once in TablesVIII-XXI and at least once in tables XXII to XLIX, or an oligonucleotidethat encodes the HLA peptide.

Another embodiment of the invention is antibody epitopes, which comprisea peptide regions, or an oligonucleotide encoding the peptide region,that has one two, three, four, or five of the following characteristics:

i) a peptide region of at least 5 amino acids of a particular peptide ofFIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a valueequal to 1.0, in the Hydrophilicity profile of FIG. 5;

ii) a peptide region of at least 5 amino acids of a particular peptideof FIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or having a value equalto 0.0, in the Hydropathicity profile of FIG. 6;

iii) a peptide region of at least 5 amino acids of a particular peptideof FIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a valueequal to 1.0, in the Percent Accessible Residues profile of FIG. 7;

iv) a peptide region of at least 5 amino acids of a particular peptideof FIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a valueequal to 1.0, in the Average Flexibility profile of FIG. 8; or

v) a peptide region of at least 5 amino acids of a particular peptide ofFIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a valueequal to 1.0, in the Beta-turn profile of FIG. 9.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The 24P4C12 SSH sequence of 160 nucleotides.

FIG. 2. A) The cDNA and amino acid sequence of 24P4C12 variant 1 (alsocalled “24P4C12 v.1” or “24P4C12 variant 1”) is shown in FIG. 2A. Thestart methionine is underlined. The open reading frame extends fromnucleic acid 6-2138 including the stop codon.

B) The cDNA and amino acid sequence of 24P4C12 variant 2 (also called“24P4C12 v.2”) is shown in FIG. 2B. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 6-2138including the stop codon.

C) The cDNA and amino acid sequence of 24P4C12 variant 3 (also called“24P4C12 v.3”) is shown in FIG. 2C. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 6-2138including the stop codon.

D) The cDNA and amino acid sequence of 24P4C12 variant 4 (also called“24P4C12 v.4”) is shown in FIG. 2D. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 6-2138including the stop codon.

E) The cDNA and amino acid sequence of 24P4C12 variant 5 (also called“24P4C12 v.5”) is shown in FIG. 2E. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 6-2138including the stop codon.

F) The cDNA and amino acid sequence of 24P4C12 variant 6 (also called“24P4C12 v.6”) is shown in FIG. 2F. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 6-2138including the stop codon.

G) The cDNA and amino acid sequence of 24P4C12 variant 7 (also called“24P4C12 v.7”) is shown in FIG. 2G. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 6-1802including the stop codon.

H) The cDNA and amino acid sequence of 24P4C12 variant 8 (also called“24P4C12 v.8”) is shown in FIG. 2H. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 6-2174including the stop codon.

I) The cDNA and amino acid sequence of 24P4C12 variant 9 (also called“24P4612 v.9”) is shown in FIG. 2I. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 6-2144including the stop codon.

FIG. 3.

A) Amino acid sequence of 24P4C12 v.1 is shown in FIG. 3A; it has 710amino acids.

B) The amino acid sequence of 24P4C12 v.3 is shown in FIG. 3B; it has710 amino acids.

C) The amino acid sequence of 24P4C12 v.5 is shown in FIG. 3C; it has710 amino acids.

D) The amino acid sequence of 24P4C12 v.6 is shown in FIG. 3D; it has710 amino acids.

E) The amino acid sequence of 24P4C12 v.7 is shown in FIG. 3E; it has598 amino acids.

F) The amino acid sequence of 24P4C12 v.8 is shown in FIG. 3F; it has722 amino acids.

G) The amino acid sequence of 24P4C12 v.9 is shown in FIG. 3G; it has712 amino acids. As used herein, a reference to 24P4C12 includes allvariants thereof, including those shown in FIGS. 2, 3, 10, and 11,unless the context clearly indicates otherwise.

FIG. 4. Alignment or 24P4C12 with human choline transporter-like protein4 (CTL4) (gil 14249468).

FIG. 5. Hydrophilicity amino acid profile of 24P4C12 determined bycomputer algorithm sequence analysis using the method of Hopp and Woods(Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A.78:3824-3828) accessed on the Protscale website located on the WorldWide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasymolecular biology server.

FIG. 6. Hydropathicity amino acid profile of 24P4C12 determined bycomputer algorithm sequence analysis using the method of Kyte andDoolittle (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132)accessed on the ProtScale website located on the World Wide Web at(.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biologyserver.

FIG. 7. Percent accessible residues amino acid profile of 24P4C12determined by computer algorithm sequence analysis using the method ofJanin (Janin J., 1979 Nature 277:491-492) accessed on the ProtScalewebsite located on the World Wide Web at(.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biologyserver.

FIG. 8. Average flexibility amino acid profile of 24P4C12 determined bycomputer algorithm sequence analysis using the method of Bhaskaran andPonnuswamy (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept.Protein Res. 32:242-255) accessed on the ProtScale website located onthe World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through theExPasy molecular biology server.

FIG. 9. Beta-turn amino acid profile of 24P4C12 determined by computeralgorithm sequence analysis using the method of Deleage and Roux(Deleage, G., Roux B. 1987 Protein Engineering 1:289-294) accessed onthe ProtScale website located on the World Wide Web at(.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biologyserver.

FIG. 10. Schematic alignment of SNP variants of 24P4C12. Variants24P4C12 v.2 through v.6 are variants with single nucleotide differences.Though these SNP variants are shown separately, they could also occur inany combinations and in any transcript variants that contains the basepairs. Numbers correspond to those of 24P4C12 v.1. Black box shows thesame sequence as 24P4C12 v.1. SNPs are indicated above the box.

FIG. 11. Schematic alignment of protein variants of 24P4C12. Proteinvariants correspond to nucleotide variants. Nucleotide variants 24P4C12v.2, v.4 in FIG. 10 code for the same protein as 24P4C12 v.1. Nucleotidevariants 24P4C12 v.7, v.8 and v.9 are splice variants of v.1, as shownin FIG. 12. Single amino acid differences were indicated above theboxes. Black boxes represent the same sequence as 24P4C12 v.1. Numbersunderneath the box correspond to 24P4C12 v.1.

FIG. 12. Exon compositions of transcript variants of 24P4C12. Variant24P4C12 v.7, v.8 and v.9 are transcript variants of 24P4C12 v.1. Variant24P4C12 v.7 does not have exons 10 and 11 of variant 24P4C12 v.1.Variant 24P4C12 v.8 extended 36 bp at the 3′ end of exon 20 of variant24P4C12 v.1. Variant 24P4C12 v.9 had a longer exon 12 and shorter exon13 as compared to variant 24P4C12v.1. Numbers in “( )” underneath theboxes correspond to those of 24P4C12v.1. Lengths of introns and exonsare not proportional.

FIG. 13. Secondary structure and transmembrane domains prediction for24P4C12 protein variant 1 (SEQ ID NO: 112). A: The secondary structureof 24P4C12 protein variant 1 was predicted using the HNN—HierarchicalNeural Network method (Guermeur, 1997,http://pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html), accessedfrom the ExPasy molecular biology server (http://www.expasy.ch/tools/).This method predicts the presence and location of alpha helices,extended strands, and random coils from the primary protein sequence.The percent of the protein in a given secondary structure is alsolisted. B: Schematic representation of the probability of existence oftransmembrane regions and orientation of 24P4C12 variant 1 based on theTMpred algorithm of Hofmann and Stoffel which utilizes TMBASE (K.Hofmann, W. Stoffel. TMBASE—A database of membrane spanning proteinsegments Biol. Chem. Hoppe-Seyler 374:166, 1993). C: Schematicrepresentation of the probability of the existence of transmembraneregions and the extracellular and intracellular orientation of 24P4C12variant 1 based on the TMHMM algorithm of Sonnhammer, von Heijne, andKrogh (Erik L. L. Sonnhammer, Gunnar von Heijne, and Anders Krogh: Ahidden Markov model for predicting transmembrane helices in proteinsequences. In Proc. of Sixth Int. Conf. on Intelligent Systems forMolecular Biology, p 175-182 Ed J. Glasgow, T. Littlejohn, F. Major, R.Lathrop, D. Sankoff, and C. Sensen Menlo Park, Calif.: AAAI Press,1998). The TMpred and TMHMM algorithms are accessed from the ExPasymolecular biology server (http://www.expasy.ch/tools/).

FIG. 14. 24P4C12 Expression by RT-PCR. First strand cDNA was generatedfrom vital pool 1 (kidney, liver and lung), vital pool 2 (colon,pancreas and stomach), a pool of prostate cancer xenografts (LAPC-4AD,LAPC-4AI, LAPC-9AD and LAPC-9AI), prostate cancer pool, bladder cancerpool, kidney cancer pool, colon cancer pool, ovary cancer pool, breastcancer pool, and cancer metastasis pool. Normalization was performed byPCR using primers to actin. Semi-quantitative PCR, using primers to24P4C12, was performed at 26 and 30 cycles of amplification. Resultsshow strong expression of 24P4C12 in prostate cancer pool and ovarycancer pool. Expression was also detected in prostate cancer xenografts,bladder cancer pool, kidney cancer pool, colon cancer pool, breastcancer pool, cancer metastasis pool, vital pool 1, and vital pool 2.

FIG. 15. Expression of 24P4C12 in normal tissues. Two multiple tissuenorthern blots (Clontech) both with 2 ug of mRNA/lane were probed withthe 24P4C12 sequence. Size standards in kilobases (kb) are indicated onthe side. Results show expression of 24P4C12 in prostate, kidney andcolon. Lower expression is detected in pancreas, lung and placentaamongst all 16 normal tissues tested.

FIG. 16. Expression of 24P4C12 in Prostate Cancer Xenografts and CellLines. RNA was extracted from a panel of cell lines and prostate cancerxenografts (PrEC, LAPC-4AD, LAPC-4AI, LAPC-9AD, LAPC-9AI, LNCaP, PC-3,DU145, TsuPr, and LAPC-4CL). Northern blot with 10 ug of total RNA/lanewas probed with 24P4C12 SSH sequence. Size standards in kilobases (kb)are indicated on the side. The 24P4C12 transcript was detected inLAPC-4AD, LAPC-4AI, LAPC-9AD, LAPC-9AI, LNCaP, and LAPC-4 CL.

FIG. 17. Expression of 24P4C12 in Patient Cancer Specimens and NormalTissues. RNA was extracted from a pool of prostate cancer specimens,bladder cancer specimens, colon cancer specimens, ovary cancerspecimens, breast cancer specimens and cancer metastasis specimens, aswell as from normal prostate (NP), normal bladder (NB), normal kidney(NK), and normal colon (NC). Northern blot with 10 μg of total RNA/lanewas probed with 24P4C12 SSH sequence. Size standards in kilobases (kb)are indicated on the side. Strong expression of 24P4C12 transcript wasdetected in the patient cancer pool specimens, and in normal prostatebut not in the other normal tissues tested.

FIG. 18. Expression of 24P4C12 in Prostate Cancer Patient Specimens. RNAwas extracted from normal prostate (N), prostate cancer patient tumors(T) and their matched normal adjacent tissues (Nat). Northern blots with10 ug of total RNA were probed with the 24P4C12 SSH fragment. Sizestandards in kilobases are on the side. Results show expression of24P4C12 in normal prostate and all prostate patient tumors tested.

FIG. 19. Expression of 24P4C12 in Colon Cancer Patient Specimens. RNAwas extracted from colon cancer cell lines (CL: Colo 205, LoVo, andSK—CO—), normal colon (N), colon cancer patient tumors (T) and theirmatched normal adjacent tissues (Nat). Northern blots with 10 ug oftotal RNA were probed with the 24P4C12 SSH fragment. Size standards inkilobases are on the side. Results show expression of 24P4C12 in normalcolon and all colon patient tumors tested. Expression was detected inthe cell lines Colo 205 and SK—CO—, but not in LoVo.

FIG. 20. Expression of 24P4C12 in Lung Cancer Patient Specimens. RNA wasextracted from lung cancer cell lines (CL: CALU-1, A427, NCI-H82,NCI-H146), normal lung (N), lung cancer patient tumors (T) and theirmatched normal adjacent tissues (Nat). Northern blots with 10 ug oftotal RNA were probed with the 24P4C12 SSH fragment. Size standards inkilobases are on the side. Results show expression of 24P4C12 in lungpatient tumors tested, but not in normal lung. Expression was alsodetected in CALU-1, but not in the other cell lines A427, NCI-H82, andNCI-H1 46.

FIG. 21. Expression of 24P4C12 in breast and stomach human cancerspecimens. Expression of 24P4C12 was assayed in a panel of human stomachand breast cancers (T) and their respective matched normal tissues (N)on RNA dot blots. 24P4C12 expression was seen in both stomach and breastcancers. The expression detected in normal adjacent tissues (isolatedfrom diseased tissues) but not in normal tissues (isolated from healthydonors) may indicate that these tissues are not fully normal and that24P4C12 may be expressed in early stage tumors.

FIG. 22. 24P4C12 Expression in a large panel of Patient CancerSpecimens. First strand cDNA was prepared from a panel of ovary patientcancer specimens (A), uterus patient cancer specimens (B), prostatecancer specimens (C), bladder cancer patient specimens (D), lung cancerpatient specimens (E), pancreas cancer patient specimens (F), coloncancer specimens (G), and kidney cancer specimens (H). Normalization wasperformed by PCR using primers to actin. Semi-quantitative PCR, usingprimers to 24P4C12, was performed at 26 and 30 cycles of amplification.Samples were run on an agarose gel, and PCR products were quantitatedusing the AlphaImager software. Expression was recorded as absent, low,medium or strong. Results show expression of 24P4C12 in the majority ofpatient cancer specimens tested, 73.3% of ovary patient cancerspecimens, 83.3% of uterus patient cancer specimens, 95.0% of prostatecancer specimens, 61.1% of bladder cancer patient specimens, 80.6% oflung cancer patient specimens, 87.5% of pancreas cancer patientspecimens, 87.5% of colon cancer specimens, 68.4% of clear cell renalcarcinoma, 100% of papillary renal cell carcinoma.

FIG. 23. 24P4C12 expression in transduced cells. PC3 prostate cancercells, NIH-3T3 mouse cells and 300.19 mouse cells were transduced with24P4C12.pSRa retroviral vector. Cells were selected in neomycin for thegeneration of stable cell lines. RNA was extracted following selectionin neomycin. Northern blots with 10 ug of total RNA were probed with the24P4C12 SSH fragment. Results show strong expression of 24P4C12 in24P4C12.pSRa transduced PC3, 3T3 and 300.19 cells, but not in thecontrol cells transduced with the parental pSRa construct.

FIG. 24. Expression of 24P4C12 in 293T cells. 293T cell were transientlytransfected with either pcDNA3.1 Myc-His tagged expression vector, thepSR

expression vector each encoding the 24P4C12 variant 1 cDNA or a controlneo vector. Cells were harvested 2 days later and analyzed by Westernblot with anti-24P4C12 pAb (A) or by Flow cytometry (B) on fixed andpermeabilized 293T cells with either the anti-24P4C12 pAb or anti-HispAb followed by a PE-conjugated anti-rabbit IgG secondary Ab. Shown isexpression of the monomeric and aggregated forms of 24P4C12 by Westernblot and a fluorescent shift of 24P4C12-293T cells compared to controlneo cells when stained with the anti-24P4C12 and anti-His pAbs which aredirected to the intracellular NH3 and COCH termini, respectively.

FIG. 25. Expression and detection of 24P4C12 in stably transduced PC3cells. PC3 cells were infected with retrovirus encoding the 24P4C12variant 1 cDNA and stably transduced cells were derived by G418selection. Cells were then analyzed by Western blot (A) orimmunohistochemistry (B) with anti-24P4C12 pAb. Shown with an arrow onthe Western blot is expression of a ˜94 kD band representing 24P4C12expressed in PC3-24P4C12 cells but not in control neo cells.Immunohistochemical analysis shows specific staining of 24P4C12-PC3cells and not PC3-neo cells which is competed away competitor peptide towhich the pAb was derived.

FIG. 26. Expression of recombinant 24P4C12 antigens in 293T cells. 293Tcells were transiently transfected with Tag5 His-tagged expressionvectors encoding either amino acids 59-227 or 319-453 of 24P4C12 variant1 or a control vector. 2 days later supernatants were collected andcells harvested and lysed. Supernatants and lysates were then subjectedto Western blot analysis using an anti-His pAb. Shown is expression ofthe recombinant Tag5 59-227 protein in both the supernatant and lysateand the Tag5 319-453 protein in the cell lysate. These proteins arepurified and used as antigens for generation of 24P4C12-specificantibodies.

FIG. 27. Monoclonal antibodies detect 24P4C12 protein expression in 293Tcells by flow cytometry. 293T cells were transfected with either pCDNA3.1 His-tagged expression vector for 24P4C12 or a control neo vector andharvested 2 days later. Cells were fixed, permeabilized, and stainedwith a 1:2 dilution of supernatants of the indicated hybridomasgenerated from mice immunized with 300.19-24P4C12 cells or with anti-HispAb. Cells were then stained with a PE-conjugated secondary Ab andanalyzed by flow cytometry. Shown is a fluorescent shift of 293T-24P4C12cells but not control neo cells demonstrating specific recognition of24P4C12 protein by the hybridoma supernatants.

FIG. 28. Shows expression of 24P4C12 Enhances Proliferation. PC3 and 3T3were grown overnight in low FBS. Cells were then incubated in low or 10%FBS as indicated. Proliferation was measured by Alamar Blue.

FIG. 29. Detection of 24P4C12 protein by immunohistochemistry inprostate cancer patient specimens. Prostate adenocarcinoma tissue andits matched normal adjacent tissue were obtained from prostate cancerpatients. The results showed strong expression of 24P4C12 in the tumorcells and normal epithelium of the prostate cancer patients' tissue(panels (A) low grade prostate adenocarcinoma, (B) high grade prostateadenocarcinoma, (C) normal tissue adjacent to tumor). The expression wasdetected mostly around the cell membrane indicating that 24P4C12 ismembrane associated in prostate tissues.

FIG. 30. Detection of 24P4C12 protein by immunohistochemistry in variouscancer patient specimens. Tissue was obtained from patients with colonadenocarcinoma, breast ductal carcinoma, lung adenocarcinoma, bladdertransitional cell carcinoma, renal clear cell carcinoma and pancreaticadenocarcinoma. The results showed expression of 24P4C12 in the tumorcells of the cancer patients' tissue (panel (A) colon adenocarcinoma,(B) lung adenocarcinoma, (C) breast ductal carcinoma, (D) bladdertransitional carcinoma, (E) renal clear cell carcinoma, (F) pancreaticadenocarcinoma).

FIG. 31. Shows 24P4C12 Enhances Tumor Growth in SCID Mice. 1×106PC3-24P4C12 cells were mixed with Matrigel and injected on the right andleft subcutaneous flanks of 4 male SCID mice per group. Each data pointrepresents mean tumor volume (n=8).

FIG. 32. Shows 24P4C12 Enhances Tumor Growth in SCID Mice. 1×1063T3-24P4C12 cells were mixed with Matrigel and injected on the rightsubcutaneous flanks of 7 male SCID mice per group. Each data pointrepresents mean tumor volume (n=6).

DETAILED DESCRIPTION OF THE INVENTION

Outline of Sections

I.) Definitions

II.) 24P4C12 Polynucleotides

II.A.) Uses of 24P4C12 Polynucleotides

II.A.1.) Monitoring of Genetic Abnormalities

II.A.2.) Antisense Embodiments

II.A.3.) Primers and Primer Pairs

-   -   II.A.4.) Isolation of 24P4C12-Encoding Nucleic Acid Molecules

II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems

III.) 24P4C12-related Proteins

-   -   III.A.) Motif-bearing Protein Embodiments    -   III.B.) Expression of 24P4C12-related Proteins    -   III.C.) Modifications of 24P4C12-related Proteins    -   III.D.) Uses of 24P4C12-related Proteins

IV.) 24P4C12 Antibodies

V.) 24P4C12 Cellular Immune Responses

VI.) 24P4C12 Transgenic Animals

VII.) Methods for the Detection of 24P4C12

VIII.) Methods for Monitoring the Status of 24P4C12-related Genes andTheir Products

IX.) Identification of Molecules That Interact With 24P4C12

X.) Therapeutic Methods and Compositions

-   -   X.A.) Anti-Cancer Vaccines

X.B.) 24P4C12 as a Target for Antibody-Based Therapy

X.C.) 24P4C12 as a Target for Cellular Immune Responses

-   -   X.C.1. Minigene Vaccines    -   X.C.2. Combinations of CTL Peptides with Helper Peptides    -   X.C.3. Combinations of CTL Peptides with T Cell Priming Agents    -   X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or        HTL Peptides    -   X.D.) Adoptive Immunotherapy

X.E.) Administration of Vaccines for Therapeutic or ProphylacticPurposes

XI.) Diagnostic and Prognostic Embodiments of 24P4C12.

XII.) Inhibition of 24P4C12 Protein Function

-   -   XII.A.) Inhibition of 24P4C12 With Intracellular Antibodies    -   XII.B.) Inhibition of 24P4C12 with Recombinant Proteins    -   XII.C.) Inhibition of 24P4C12 Transcription or Translation    -   XII.D.) General Considerations for Therapeutic Strategies

XIII.) Identification, Characterization and Use of Modulators of 24P4C12

XIV.) KITS/Articles of Manufacture

I.) DEFINITIONS

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Asappropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturerdefined protocols and/or parameters unless otherwise noted.

The terms “advanced prostate cancer”, “locally advanced prostatecancer”, “advanced disease” and “locally advanced disease” mean prostatecancers that have extended through the prostate capsule, and are meantto include stage C disease under the American Urological Association(AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, andstage T3-T4 and N+ disease under the TNM (tumor, node, metastasis)system. In general, surgery is not recommended for patients with locallyadvanced disease, and these patients have substantially less favorableoutcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

“Altering the native glycosylation pattern” is intended for purposesherein to mean deleting one or more carbohydrate moieties found innative sequence 24P4C12 (either by removing the underlying glycosylationsite or by deleting the glycosylation by chemical and/or enzymaticmeans), and/or adding one or more glycosylation sites that are notpresent in the native sequence 24P4C12. In addition, the phrase includesqualitative changes in the glycosylation of the native proteins,involving a change in the nature and proportions of the variouscarbohydrate moieties present.

The term “analog” refers to a molecule which is structurally similar orshares similar or corresponding attributes with another molecule (e.g. a24P4C12-related protein). For example, an analog of a 24P4C12 proteincan be specifically bound by an antibody or T cell that specificallybinds to 24P4C12.

The term “antibody” is used in the broadest sense. Therefore, an“antibody” can be naturally occurring or man-made such as monoclonalantibodies produced by conventional hybridoma technology. Anti-24P4C12antibodies comprise monoclonal and polyclonal antibodies as well asfragments containing the antigen-binding domain and/or one or morecomplementarity determining regions of these antibodies.

An “antibody fragment” is defined as at least a portion of the variableregion of the immunoglobulin molecule that binds to its target, i.e.,the antigen-binding region. In one embodiment it specifically coverssingle anti-24P4C12 antibodies and clones thereof (including agonist,antagonist and neutralizing antibodies) and anti-24P4C12 antibodycompositions with polyepitopic specificity.

The term “codon optimized sequences” refers to nucleotide sequences thathave been optimized for a particular host species by replacing anycodons having a usage frequency of less than about 20%. Nucleotidesequences that have been optimized for expression in a given hostspecies by elimination of spurious polyadenylation sequences,elimination of exon/intron splicing signals, elimination oftransposon-like repeats and/or optimization of GC content in addition tocodon optimization are referred to herein as an “expression enhancedsequences.”

A “combinatorial library” is a collection of diverse chemical compoundsgenerated by either chemical synthesis or biological synthesis bycombining a number of chemical “building blocks” such as reagents. Forexample, a linear combinatorial chemical library, such as a polypeptide(e.g., mutein) library, is formed by combining a set of chemicalbuilding blocks called amino acids in every possible way for a givencompound length (i.e., the number of amino acids in a polypeptidecompound). Numerous chemical compounds are synthesized through suchcombinatorial mixing of chemical building blocks (Gallop et al., J. Med.Chem. 37(9): 1233-1251 (1994)).

Preparation and screening of combinatorial libraries is well known tothose of skill in the art. Such combinatorial chemical librariesinclude, but are not limited to, peptide libraries (see, e.g., U.S. Pat.No. 5,010,175, Furka, Pept. Prot. Res. 37:487-493 (1991), Houghton etal., Nature, 354:84-88 (1991)), peptoids (PCT Publication No WO91/19735), encoded peptides (PCT Publication WO 93/20242), randombio-oligomers (PCT Publication WO 92/00091), benzodiazepines (U.S. Pat.No. 5,288,514), diversomers such as hydantoins, benzodiazepines anddipeptides (Hobbs et al., Proc. Nat. Acad. Sol. USA 90:6909-6913(1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc.114:6568 (1992)), nonpeptidal peptidomimetics with a Beta-D-Glucosescaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218(1992)), analogous organic syntheses of small compound libraries (Chenet al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho, etal., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell etal., J. Org. Chem. 59:658 (1994)). See, generally, Gordon et al., J.Med. Chem. 37:1385 (1994), nucleic acid libraries (see, e.g.,Stratagene, Corp.), peptide nucleic acid libraries (see, e.g., U.S. Pat.No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., NatureBiotechnology 14(3): 309-314 (1996), and PCT/US96/10287), carbohydratelibraries (see, e.g., Liang et al., Science 274:1520-1522 (1996), andU.S. Pat. No. 5,593,853), and small organic molecule libraries (see,e.g., benzodiazepines, Baum, C&EN, January 18, page 33 (1993);isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones andmetathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos.5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337;benzodiazepines, U.S. Pat. No. 5,288,514; and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 NIPS, 390 NIPS, Advanced Chem Tech, LouisvilleKy.; Symphony, Rainin, Woburn, Mass.; 433A, Applied Biosystems, FosterCity, Calif.; 9050, Plus, Millipore, Bedford, NIA). A number ofwell-known robotic systems have also been developed for solution phasechemistries. These systems include automated workstations such as theautomated synthesis apparatus developed by Takeda Chemical Industries,LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms(Zymate H, Zymark Corporation, Hopkinton, Mass.; Orca, Hewleff-Packard,Palo Alto, Calif.), which mimic the manual synthetic operationsperformed by a chemist. Any of the above devices are suitable for usewith the present invention. The nature and implementation ofmodifications to these devices (if any) so that they can operate asdiscussed herein will be apparent to persons skilled in the relevantart. In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J.; Asinex,Moscow, R U; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd, Moscow, R U;3D Pharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md.;etc.).

The term “cytotoxic agent” refers to a substance that inhibits orprevents the expression activity of cells, function of cells and/orcauses destruction of cells. The term is intended to include radioactiveisotopes chemotherapeutic agents, and toxins such as small moleculetoxins or enzymatically active toxins of bacterial, fungal, plant oranimal origin, including fragments and/or variants thereof. Examples ofcytotoxic agents include, but are not limited to auristatins,auromycins, maytansinoids, yttrium, bismuth, ricin, ricin A-chain,combrestatin, duocarmycins, dolostatins, doxorubicin, daunorubicin,taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracindione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40,abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin,mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin,calicheamicin, Sapaonaria officinalis inhibitor, and glucocorticoid andother chemotherapeutic agents, as well as radioisotopes such as At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi^(212 or 213), P³² andradioactive isotopes of Lu including Lu¹⁷⁷. Antibodies may also beconjugated to an anti-cancer pro-drug activating enzyme capable ofconverting the pro-drug to its active form.

The “gene product” is sometimes referred to herein as a protein or mRNA.For example, a “gene product of the invention” is sometimes referred toherein as a “cancer amino acid sequence”, “cancer protein”, “protein ofa cancer listed in Table I”, a “cancer mRNA”, “mRNA of a cancer listedin Table I”, etc. In one embodiment, the cancer protein is encoded by anucleic acid of FIG. 2. The cancer protein can be a fragment, oralternatively, be the full-length protein to the fragment encoded by thenucleic acids of FIG. 2. In one embodiment, a cancer amino acid sequenceis used to determine sequence identity or similarity. In anotherembodiment, the sequences are naturally occurring allelic variants of aprotein encoded by a nucleic acid of FIG. 2. In another embodiment, thesequences are sequence variants as further described herein.

“High throughput screening” assays for the presence, absence,quantification, or other properties of particular nucleic acids orprotein products are well known to those of skill in the art. Similarly,binding assays and reporter gene assays are similarly well known. Thus,e.g., U.S. Pat. No. 5,559,410 discloses high throughput screeningmethods for proteins; U.S. Pat. No. 5,585,639 discloses high throughputscreening methods for nucleic acid binding (i.e., in arrays); while U.S.Pat. Nos. 5,576,220 and 5,541,061 disclose high throughput methods ofscreening for ligand/antibody binding.

In addition, high throughput screening systems are commerciallyavailable (see, e.g., Amersham Biosciences, Piscataway, N.J.; ZymarkCorp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio; BeckmanInstruments, Inc. Fullerton, Calif.; Precision Systems, Inc., Natick,Mass.; etc.). These systems typically automate entire procedures,including all sample and reagent pipetting, liquid dispensing, timedincubations, and final readings of the microplate in detector(s)appropriate for the assay. These configurable systems provide highthroughput and rapid start up as well as a high degree of flexibilityand customization. The manufacturers of such systems provide detailedprotocols for various high throughput systems. Thus, e.g., Zymark Corp.provides technical bulletins describing screening systems for detectingthe modulation of gene transcription, ligand binding, and the like.

The term “homolog” refers to a molecule which exhibits homology toanother molecule, by for example, having sequences of chemical residuesthat are the same or similar at corresponding positions.

“Human Leukocyte Antigen” or “HLA” is a human class I or class II MajorHistocompatibility Complex (MHC) protein (see, e.g., Stites, et al.,IMMUNOLOGY, 8^(TH) ED., Lange Publishing, Los Altos, Calif. (1994).

The terms “hybridize”, “hybridizing”, “hybridizes” and the like, used inthe context of polynucleotides, are meant to refer to conventionalhybridization conditions, preferably such as hybridization in 50%formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperatures forhybridization are above 37 degrees C. and temperatures for washing in0.1×SSC/0.1% SDS are above 55 degrees C.

The phrases “isolated” or ‘biologically pure’ refer to material which issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state. Thus,isolated peptides in accordance with the invention preferably do notcontain materials normally associated with the peptides in their in situenvironment. For example, a polynucleotide is said to be “isolated” whenit is substantially separated from contaminant polynucleotides thatcorrespond or are complementary to genes other than the 24P4C12 genes orthat encode polypeptides other than 24P4C12 gene product or fragmentsthereof. A skilled artisan can readily employ nucleic acid isolationprocedures to obtain an isolated 24P4C12 polynucleotide. A protein issaid to be “isolated,” for example, when physical, mechanical orchemical methods are employed to remove the 24P4C12 proteins fromcellular constituents that are normally associated with the protein. Askilled artisan can readily employ standard purification methods toobtain an isolated 24P4C12 protein. Alternatively, an isolated proteincan be prepared by chemical means.

The term “mammal” refers to any organism classified as a mammal,including mice, rats, rabbits, dogs, cats, cows, horses and humans. Inone embodiment of the invention, the mammal is a mouse. In anotherembodiment of the invention, the mammal is a human.

The terms “metastatic prostate cancer” and “metastatic disease” meanprostate cancers that have spread to regional lymph nodes or to distantsites, and are meant to include stage D disease under the AUA system andstage T×N×M+ under the TNM system. As is the case with locally advancedprostate cancer, surgery is generally not indicated for patients withmetastatic disease, and hormonal (androgen ablation) therapy is apreferred treatment modality. Patients with metastatic prostate cancereventually develop an androgen-refractory state within 12 to 18 monthsof treatment initiation. Approximately half of these androgen-refractorypatients die within 6 months after developing that status. The mostcommon site for prostate cancer metastasis is bone. Prostate cancer bonemetastases are often osteoblastic rather than osteolytic (i.e.,resulting in net bone formation). Bone metastases are found mostfrequently in the spine, followed by the femur, pelvis, rib cage, skulland humerus. Other common sites for metastasis include lymph nodes,lung, liver and brain. Metastatic prostate cancer is typically diagnosedby open or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy.

The term “modulator” or “test compound” or “drug candidate” orgrammatical equivalents as used herein describe any molecule, e.g.,protein, oligopeptide, small organic molecule, polysaccharide,polynucleotide, etc., to be tested for the capacity to directly orindirectly alter the cancer phenotype or the expression of a cancersequence, e.g., a nucleic acid or protein sequences, or effects ofcancer sequences (e.g., signaling, gene expression, protein interaction,etc.) In one aspect, a modulator will neutralize the effect of a cancerprotein of the invention. By “neutralize” is meant that an activity of aprotein is inhibited or blocked, along with the consequent effect on thecell. In another aspect, a modulator will neutralize the effect of agene, and its corresponding protein, of the invention by normalizinglevels of said protein. In preferred embodiments, modulators alterexpression profiles, or expression profile nucleic acids or proteinsprovided herein, or downstream effector pathways. In one embodiment, themodulator suppresses a cancer phenotype, e.g. to a normal tissuefingerprint. In another embodiment, a modulator induced a cancerphenotype. Generally, a plurality of assay mixtures is run in parallelwith different agent concentrations to obtain a differential response tothe various concentrations. Typically, one of these concentrationsserves as a negative control, i.e., at zero concentration or below thelevel of detection.

Modulators, drug candidates or test compounds encompass numerouschemical classes, though typically they are organic molecules,preferably small organic compounds having a molecular weight of morethan 100 and less than about 2,500 Daltons. Preferred small moleculesare less than 2000, or less than 1500 or less than 1000 or less than 500D. Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Modulators also comprise biomolecules such aspeptides, saccharides, fatty acids, steroids, purines, pyrimidines,derivatives, structural analogs or combinations thereof. Particularlypreferred are peptides. One class of modulators are peptides, forexample of from about five to about 35 amino acids, with from about fiveto about 20 amino acids being preferred, and from about 7 to about 15being particularly preferred. Preferably, the cancer modulatory proteinis soluble, includes a non-transmembrane region, and/or, has anN-terminal Cys to aid in solubility. In one embodiment, the C-terminusof the fragment is kept as a free acid and the N-terminus is a freeamine to aid in coupling, i.e., to cysteine. In one embodiment, a cancerprotein of the invention is conjugated to an immunogenic agent asdiscussed herein. In one embodiment, the cancer protein is conjugated toBSA. The peptides of the invention, e.g., of preferred lengths, can belinked to each other or to other amino acids to create a longerpeptide/protein. The modulatory peptides can be digests of naturallyoccurring proteins as is outlined above, random peptides, or “biased”random peptides. In a preferred embodiment, peptide/protein-basedmodulators are antibodies, and fragments thereof, as defined herein.

Modulators of cancer can also be nucleic acids. Nucleic acid modulatingagents can be naturally occurring nucleic acids, random nucleic acids,or “biased” random nucleic acids. For example, digests of prokaryotic oreukaryotic genomes can be used in an approach analogous to that outlinedabove for proteins.

The term “monoclonal antibody” refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the antibodiescomprising the population are identical except for possible naturallyoccurring mutations that are present in minor amounts.

A “motif”, as in biological motif of a 24P4C12-related protein, refersto any pattern of amino acids forming part of the primary sequence of aprotein, that is associated with a particular function (e.g.protein-protein interaction, protein-DNA interaction, etc) ormodification (e.g. that is phosphorylated, glycosylated or amidated), orlocalization (e.g. secretory sequence, nuclear localization sequence,etc.) or a sequence that is correlated with being immunogenic, eitherhumorally or cellularly. A motif can be either contiguous or capable ofbeing aligned to certain positions that are generally correlated with acertain function or property. In the context of HLA motifs, “motif”refers to the pattern of residues in a peptide of defined length,usually a peptide of from about 8 to about 13 amino acids for a class IHLA motif and from about 6 to about 25 amino acids for a class II HLAmotif, which is recognized by a particular HLA molecule. Peptide motifsfor HLA binding are typically different for each protein encoded by eachhuman HLA allele and differ in the pattern of the primary and secondaryanchor residues.

A “pharmaceutical excipient” comprises a material such as an adjuvant, acarrier, pH-adjusting and buffering agents, tonicity adjusting agents,wetting agents, preservative, and the like.

“Pharmaceutically acceptable” refers to a non-toxic, inert, and/orcomposition that is physiologically compatible with humans or othermammals.

The term “polynucleotide” means a polymeric form of nucleotides of atleast 10 bases or base pairs in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide, and ismeant to include single and double stranded forms of DNA and/or RNA. Inthe art, this term if often used interchangeably with “oligonucleotide”.A polynucleotide can comprise a nucleotide sequence disclosed hereinwherein thymidine (T), as shown for example in FIG. 2, can also beuracil (U); this definition pertains to the differences between thechemical structures of DNA and RNA, in particular the observation thatone of the four major bases in RNA is uracil (U) instead of thymidine(T).

The term “polypeptide” means a polymer of at least about 4, 5, 6, 7, or8 amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used. In the art, thisterm is often used interchangeably with “peptide” or “protein”.

An HLA “primary anchor residue” is an amino acid at a specific positionalong a peptide sequence which is understood to provide a contact pointbetween the immunogenic peptide and the HLA molecule. One to three,usually two, primary anchor residues within a peptide of defined lengthgenerally defines a “motif” for an immunogenic peptide. These residuesare understood to fit in close contact with peptide binding groove of anHLA molecule, with their side chains buried in specific pockets of thebinding groove. In one embodiment, for example, the primary anchorresidues for an HLA class I molecule are located at position 2 (from theamino terminal position) and at the carboxyl terminal position of a 8,9, 10, 11, or 12 residue peptide epitope in accordance with theinvention. Alternatively, in another embodiment, the primary anchorresidues of a peptide binds an HLA class II molecule are spaced relativeto each other, rather than to the termini of a peptide, where thepeptide is generally of at least 9 amino acids in length. The primaryanchor positions for each motif and supermotif are set forth in TableIV. For example, analog peptides can be created by altering the presenceor absence of particular residues in the primary and/or secondary anchorpositions shown in Table IV. Such analogs are used to modulate thebinding affinity and/or population coverage of a peptide comprising aparticular HLA motif or supermotif.

“Radioisotopes” include, but are not limited to the following(non-limiting exemplary uses are also set forth):

Examples of Medical Isotopes:

Isotope Description of use Actinium-225 See Thorium-229 (Th-229)(AC-225) Actinium-227 Parent of Radium-223 (Ra-223) which is an alphaemitter used to treat metastases in the (AC-227) skeleton resulting fromcancer (i.e., breast and prostate cancers), and cancerradioimmunotherapy Bismuth-212 See Thorium-228 (Th-228) (Bi-212)Bismuth-213 See Thorium-229 (Th-229) (Bi-213) Cadmium-109 Cancerdetection (Cd-109) Cobalt-60 Radiation source for radiotherapy ofcancer, for food irradiators, and for sterilization of (Co-60) medicalsupplies Copper-64 A positron emitter used for cancer therapy and SPECTimaging (Cu-64) Copper-67 Beta/gamma emitter used in cancerradioimmunotherapy and diagnostic studies (i.e., breast (Cu-67) andcolon cancers, and lymphoma) Dysprosium-166 Cancer radioimmunotherapy(Dy-166) Erbium-169 Rheumatoid arthritis treatment, particularly for thesmall joints associated with fingers and (Er-69) toes Europium-152Radiation source for food irradiation and for sterilization of medicalsupplies (Eu-152) Europium-154 Radiation source for food irradiation andfor sterilization of medical supplies (Eu-154) Gadolinium-153Osteoporosis detection and nuclear medical quality assurance devices(Gd-153) Gold-198 Implant and intracavity therapy of ovarian, prostate,and brain cancers (Au-198) Holmium-166 Multiple myeloma treatment intargeted skeletal therapy, cancer radioimmunotherapy, bone (Ho-166)marrow ablation, and rheumatoid arthritis treatment Iodine-125Osteoporosis detection, diagnostic imaging, tracer drugs, brain cancertreatment, (I-125) radiolabeling, tumor imaging, mapping of receptors inthe brain, interstitial radiation therapy, brachytherapy for treatmentof prostate cancer, determination of glomerular filtration rate (GFR),determination of plasma volume, detection of deep vein thrombosis of thelegs Iodine-131 Thyroid function evaluation, thyroid disease detection,treatment of thyroid cancer as well as (I-131) other non-malignantthyroid diseases (i.e., Graves disease, goiters, and hyperthyroidism),treatment of leukemia, lymphoma, and other forms of cancer (e.g., breastcancer) using radioimmunotherapy Iridium-192 Brachytherapy, brain andspinal cord tumor treatment, treatment of blocked arteries (i.e.,(Ir-192) arteriosclerosis and restenosis), and implants for breast andprostate tumors Lutetium-177 Cancer radioimmunotherapy and treatment ofblocked arteries (i.e., arteriosclerosis and (Lu-177) restenosis)Molybdenum-99 Parent of Technetium-99m (Tc-99m) which is used forimaging the brain, liver, lungs, heart, (Mo-99) and other organs.Currently, Tc-99m is the most widely used radioisotope used fordiagnostic imaging of various cancers and diseases involving the brain,heart, liver, lungs; also used in detection of deep vein thrombosis ofthe legs Osmium-194 Cancer radioimmunotherapy (Os-194) Palladium-103Prostate cancer treatment (Pd-103) Platinum-195m Studies onbiodistribution and metabolism of cisplatin, a chemotherapeutic drug(Pt-195m) Phosphorus-32 Polycythemia rubra vera (blood cell disease) andleukemia treatment, bone cancer (P-32) diagnosis/treatment; colon,pancreatic, and liver cancer treatment; radiolabeling nucleic acids forin vitro research, diagnosis of superficial tumors, treatment of blockedarteries (i.e., arteriosclerosis and restenosis), and intracavitytherapy Phosphorus-33 Leukemia treatment, bone diseasediagnosis/treatment, radiolabeling, and treatment of (P-33) blockedarteries (i.e., arteriosclerosis and restenosis) Radium-223 SeeActinium-227 (Ac-227) (Ra-223) Rhenium-186 Bone cancer pain relief,rheumatoid arthritis treatment, and diagnosis and treatment of (Re-186)lymphoma and bone, breast, colon, and liver cancers usingradioimmunotherapy Rhenium-188 Cancer diagnosis and treatment usingradioimmunotherapy, bone cancer pain relief, (Re-188) treatment ofrheumatoid arthritis, and treatment of prostate cancer Rhodium-105Cancer radioimmunotherapy (Rh-105) Samarium-145 Ocular cancer treatment(Sm-145) Samarium-153 Cancer radioimmunotherapy and bone cancer painrelief (Sm-153) Scandium-47 Cancer radioimmunotherapy and bone cancerpain relief (Sc-47) Selenium-75 Radiotracer used in brain studies,imaging of adrenal cortex by gamma-scintigraphy, lateral (Se-75)locations of steroid secreting tumors, pancreatic scanning, detection ofhyperactive parathyroid glands, measure rate of bile acid loss from theendogenous pool Strontium-85 Bone cancer detection and brain scans(Sr-85) Strontium-89 Bone cancer pain relief, multiple myelomatreatment, and osteoblastic therapy (Sr-89) Technetium-99m SeeMolybdenum-99 (Mo-99) (Tc-99m) Thorium-228 Parent of Bismuth-212(Bi-212) which is an alpha emitter used in cancer radioimmunotherapy(Th-228) Thorium-229 Parent of Actinium-225 (Ac-225) and grandparent ofBismuth-213 (Bi-213) which are alpha (Th-229) emitters used in cancerradioimmunotherapy Thulium-170 Gamma source for blood irradiators,energy source for implanted medical devices (Tm-170) Tin-117m Cancerimmunotherapy and bone cancer pain relief (Sn-117m) Tungsten-188 Parentfor Rhenium-188 (Re-188) which is used for cancer diagnostics/treatment,bone (W-188) cancer pain relief, rheumatoid arthritis treatment, andtreatment of blocked arteries (i.e., arteriosclerosis and restenosis)Xenon-127 Neuroimaging of brain disorders, high resolution SPECTstudies, pulmonary function tests, (Xe-127) and cerebral blood flowstudies Ytterbium-175 Cancer radioimmunotherapy (Yb-175) Yttrium-90Microseeds obtained from irradiating Yttrium-89 (Y-89) for liver cancertreatment (Y-90) Yttrium-91 A gamma-emitting label for Yttrium-90 (Y-90)which is used for cancer radioimmunotherapy (Y-91) (i.e., lymphoma,breast, colon, kidney, lung, ovarian, prostate, pancreatic, andinoperable liver cancers)

By “randomized” or grammatical equivalents as herein applied to nucleicacids and proteins is meant that each nucleic acid and peptide consistsof essentially random nucleotides and amino acids, respectively. Theserandom peptides (or nucleic acids, discussed herein) can incorporate anynucleotide or amino acid at any position. The synthetic process can bedesigned to generate randomized proteins or nucleic acids, to allow theformation of all or most of the possible combinations over the length ofthe sequence, thus forming a library of randomized candidate bicactiveproteinaceous agents.

In one embodiment, a library is “fully randomized,” with no sequencepreferences or constants at any position. In another embodiment, thelibrary is a “biased random” library. That is, some positions within thesequence either are held constant, or are selected from a limited numberof possibilities. For example, the nucleotides or amino acid residuesare randomized within a defined class, e.g., of hydrophobic amino acids,hydrophilic residues, sterically biased (either small or large)residues, towards the creation of nucleic acid binding domains, thecreation of cysteines, for cross-linking, prolines for SH-3 domains,serines, threonines, tyrosines or histidines for phosphorylation sites,etc., or to purines, etc.

A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that hasbeen subjected to molecular manipulation in vitro.

Non-limiting examples of small molecules include compounds that bind orinteract with 24P4C12, ligands including hormones, neuropeptides,chemokines, odorants, phospholipids, and functional equivalents thereofthat bind and preferably inhibit 24P4C12 protein function. Suchnon-limiting small molecules preferably have a molecular weight of lessthan about 10 kDa, more preferably below about 9, about 8, about 7,about 6, about 5 or about 4 kDa. In certain embodiments, small moleculesphysically associate with, or bind, 24P4C12 protein; are not found innaturally occurring metabolic pathways; and/or are more soluble inaqueous than non-aqueous solutions

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured nucleic acidsequences to reanneal when complementary strands are present in anenvironment below their melting temperature. The higher the degree ofdesired homology between the probe and hybridizable sequence, the higherthe relative temperature that can be used. As a result, it follows thathigher relative temperatures would tend to make the reaction conditionsmore stringent, while lower temperatures less so. For additional detailsand explanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, are identified by, but not limited to, those that: (1) employlow ionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ during hybridization a denaturing agent, such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C. “Moderately stringent conditions” are described by, but not limitedto, those in Sambrook et al., Molecular Cloning: A Laboratory Manual,New York: Cold Spring Harbor Press, 1989, and include the use of washingsolution and hybridization conditions (e.g., temperature, ionic strengthand % SDS) less stringent than those described above. An example ofmoderately stringent conditions is overnight incubation at 37° C. in asolution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA,followed by washing the filters in 1×SSC at about 37-50° C. The skilledartisan will recognize how to adjust the temperature, ionic strength,etc. as necessary to accommodate factors such as probe length and thelike.

An HLA “supermotif” is a peptide binding specificity shared by HLAmolecules encoded by two or more HLA alleles. Overall phenotypicfrequencies of HLA-supertypes in different ethnic populations are setforth in Table IV (F). The non-limiting constituents of varioussupertypes are as follows:

A2: A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*6802, A*6901,A*0207

A3: A3, A11, A31, A*3301, A*6801, A*0301, A*1101, A*3101

B7: B7, B*3501-03, B*51, B*5301, B*5401, B*5501, B*5502, B*5601, B*6701,B*7801, B*0702, B*5101, B*5602

B44: B*3701, B*4402, B*4403, B*60 (B*4001), B61 (B*4006)

A1: A*0102, A*2604, A*3601, A*4301, A*8001

A24: A*24, A*30, A*2403, A*2404, A*3002, A*3003

B27: B*1401-02, B*1503, B*1509, B*1510, B*1518, B*3801-02, B*3901,B*3902, B*3903-04, B*4801-02, B*7301, B*2701-08

B58: B*1516, B*1517, B*5701, B*5702, B58

B62: B*4601, B52, B*1501 (B62), B*1502 (B75), B*1513 (B77)

Calculated population coverage afforded by different HLA-supertypecombinations are set forth in Table IV (G).

As used herein “to treat” or “therapeutic” and grammatically relatedterms, refer to any improvement of any consequence of disease, such asprolonged survival, less morbidity, and/or a lessening of side effectswhich are the byproducts of an alternative therapeutic modality; fulleradication of disease is not required.

A “transgenic animal” (e.g., a mouse or rat) is an animal having cellsthat contain a transgene, which transgene was introduced into the animalor an ancestor of the animal at a prenatal, e.g., an embryonic stage. A“transgene” is a DNA that is integrated into the genome of a cell fromwhich a transgenic animal develops.

As used herein, an HLA or cellular immune response “vaccine” is acomposition that contains or encodes one or more peptides of theinvention. There are numerous embodiments of such vaccines, such as acocktail of one or more individual peptides; one or more peptides of theinvention comprised by a polyepitopic peptide; or nucleic acids thatencode such individual peptides or polypeptides, e.g., a minigene thatencodes a polyepitopic peptide. The “one or more peptides” can includeany whole unit integer from 1-150 or more, e.g., at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides ofthe invention. The peptides or polypeptides can optionally be modified,such as by lipidation, addition of targeting or other sequences. HLAclass I peptides of the invention can be admixed with, or linked to, HLAclass II peptides, to facilitate activation of both cytotoxic Tlymphocytes and helper T lymphocytes. HLA vaccines can also comprisepeptide-pulsed antigen presenting cells, e.g., dendritic cells.

The term “variant” refers to a molecule that exhibits a variation from adescribed type or norm, such as a protein that has one or more differentamino acid residues in the corresponding position(s) of a specificallydescribed protein (e.g. the 24P4C12 protein shown in FIG. 2 or FIG. 3.An analog is an example of a variant protein. Splice isoforms and singlenucleotides polymorphisms (SNPs) are further examples of variants.

The “24P4C12-related proteins” of the invention include thosespecifically identified herein, as well as allelic variants,conservative substitution variants, analogs and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined herein or readily available in the art.Fusion proteins that combine parts of different 24P4C12 proteins orfragments thereof, as well as fusion proteins of a 24P4C12 protein and aheterologous polypeptide are also included. Such 24P4C12 proteins arecollectively referred to as the 24P4C12-related proteins, the proteinsof the invention, or 24P4C12. The term “24P4C12-related protein” refersto a polypeptide fragment or a 24P4C12 protein sequence of 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, ormore than 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65,70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275,300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625,650, or 664 or more amino acids.

II.) 24P4C12 POLYNUCLEOTIDES

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of a 24P4C12 gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding a 24P4C12-related protein and fragments thereof, DNA, RNA,DNA/RNA hybrid, and related molecules, polynucleotides oroligonucleotides complementary to a 24P4C12 gene or mRNA sequence or apart thereof, and polynucleotides or oligonucleotides that hybridize toa 24P4C12 gene, mRNA, or to a 24P4C12 encoding polynucleotide(collectively, “24P4C12 polynucleotides”). In all instances whenreferred to in this section, T can also be U in FIG. 2.

Embodiments of a 24P4C12 polynucleotide include: a 24P4C12polynucleotide having the sequence shown in FIG. 2, the nucleotidesequence of 24P4C12 as shown in FIG. 2 wherein T is U; at least 10contiguous nucleotides of a polynucleotide having the sequence as shownin FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotidehaving the sequence as shown in FIG. 2 where T is U. For example,embodiments of 24P4C12 nucleotides comprise, without limitation:

-   -   (I) a polynucleotide comprising, consisting essentially of, or        consisting of a sequence as shown in FIG. 2, wherein T can also        be U;    -   (II) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2A, from nucleotide        residue number 6 through nucleotide residue number 2138,        including the stop codon, wherein T can also be U;    -   (III) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2B, from nucleotide        residue number 6 through nucleotide residue number 2138,        including the stop codon, wherein T can also be U;    -   (IV) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2C, from nucleotide        residue number 6 through nucleotide residue number 2138,        including the a stop codon, wherein T can also be U;    -   (V) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2D, from nucleotide        residue number 6 through nucleotide residue number 2138,        including the stop codon, wherein T can also be U;    -   (VI) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2E, from nucleotide        residue number 6 through nucleotide residue number 2138,        including the stop codon, wherein T can also be U;    -   (VII) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2F, from nucleotide        residue number 6 through nucleotide residue number 2138,        including the stop codon, wherein T can also be U;    -   (VIII) a polynucleotide comprising, consisting essentially of,        or consisting of the sequence as shown in FIG. 2G, from        nucleotide residue number 6 through nucleotide residue number        1802, including the stop codon, wherein T can also be U;    -   (IX) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2H, from nucleotide        residue number 6 through nucleotide residue number 2174,        including the stop codon, wherein T can also be U;    -   (X) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2I, from nucleotide        residue number 6 through nucleotide residue number 2144,        including the stop codon, wherein T can also be U;    -   (XI) a polynucleotide that encodes a 24P4C12-related protein        that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%        homologous to an entire amino acid sequence shown in FIGS. 2A-I;    -   (XII) a polynucleotide that encodes a 24P4C12-related protein        that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%        identical to an entire amino acid sequence shown in FIGS. 2A-I;    -   (XIII) a polynucleotide that encodes at least one peptide set        forth in Tables VIII-XXI and XXII-XLIX;    -   (XIV) a polynucleotide that encodes a peptide region of at least        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3A-D in any whole number increment up        to 710 that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a        value greater than 0.5 in the Hydrophilicity profile of FIG. 5;    -   (XV) a polynucleotide that encodes a peptide region of at least        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3A-D in any whole number increment up        to 710 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        less than 0.5 in the Hydropathicity profile of FIG. 6;    -   (XVI) a polynucleotide that encodes a peptide region of at least        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3A-D in any whole number increment up        to 710 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12,13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a        value greater than 0.5 in the Percent Accessible Residues        profile of FIG. 7;    -   (XVII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3A-D in any whole number increment up        to 710 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Average Flexibility profile of FIG. 8;    -   (XVIII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3A-D in any whole number increment up        to 710 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Beta-turn profile of FIG. 9;    -   (XIX) a polynucleotide that encodes a peptide region of at least        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3E in any whole number increment up        to 598 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Hydrophilicity profile of FIG. 5;    -   (XX) a polynucleotide that encodes a peptide region of at least        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3E in any whole number increment up        to 598 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        less than 0.5 in the Hydropathicity profile of FIG. 6;    -   (XXI) a polynucleotide that encodes a peptide region of at least        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3E in any whole number increment up        to 598 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Percent Accessible Residues profile of        FIG. 7;    -   (XXII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3E in any whole number increment up        to 598 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Average Flexibility profile of FIG. 8;    -   (XXIII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11,12,13,14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3E in any whole number increment up        to 598 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Beta-turn profile of FIG. 9    -   (XXIV) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3F in any whole number increment up        to 722 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12,13,14,15,16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,        29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Hydrophilicity profile of FIG. 5;    -   (XXV) a polynucleotide that encodes a peptide region of at least        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3F in any whole number increment up        to 722 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        less than 0.5 in the Hydropathicity profile of FIG. 6;    -   (XXVI) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3F in any whole number increment up        to 722 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Percent Accessible Residues profile of        FIG. 7;    -   (XXVII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3F in any whole number increment up        to 722 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Average Flexibility profile of FIG. 8;    -   (XXVIII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3F in any whole number increment up        to 722 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Beta-turn profile of FIG. 9    -   (XXIX) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3G in any whole number increment up        to 712 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Hydrophilicity profile of FIG. 5;    -   (XXX) a polynucleotide that encodes a peptide region of at least        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3G in any whole number increment up        to 712 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12,13,14,15,16, 17,18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28,        29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value        less than 0.5 in the Hydropathicity profile of FIG. 6;    -   (XXXI) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3G in any whole number increment up        to 712 that includes 1,2,3,4,5,6,7,8,9,10, 11,12,13,14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,        33, 34, 35 amino acid position(s) having a value greater than        0.5 in the Percent Accessible Residues profile of FIG. 7;    -   (XXXII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3G in any whole number increment up        to 712 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Average Flexibility profile of FIG. 8;    -   (XXXIII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3G in any whole number increment up        to 712 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Beta-turn profile of FIG. 9    -   (XXXIV) a polynucleotide that is fully complementary to a        polynucleotide of any one of (I)-(XXXIII).    -   (XXXV) a peptide that is encoded by any of (I) to (XXXIII); and    -   (XXXVI) a composition comprising a polynucleotide of any of        (I)-(XXXIV) or peptide of (XXXV) together with a pharmaceutical        excipient and/or in a human unit dose form.    -   (XXXVII) a method of using a polynucleotide of any (I)-(XXXIV)        or peptide of (XXXV) or a composition of (XXXVI) in a method to        modulate a cell expressing 24P4C12,    -   (XXXVIII) a method of using a polynucleotide of any (I)-(XXXIV)        or peptide of (XXXV) or a composition of (XXXVI) in a method to        diagnose, prophylax, prognose, or treat an individual who bears        a cell expressing 24P4C12    -   (XXXIX) a method of using a polynucleotide of any (I)-(XXXIV) or        peptide of (XXXV) or a composition of (XXXVI) in a method to        diagnose, prophylax, prognose, or treat an individual who bears        a cell expressing 24P4C12, said cell from a cancer of a tissue        listed in Table I;    -   (XL) a method of using a polynucleotide of any (I)-(XXXIV) or        peptide of (XXXV) or a composition of (XXXVI) in a method to        diagnose, prophylax, prognose, or treat a cancer;    -   (XLI) a method of using a polynucleotide of any (I)-(XXXIV) or        peptide of (XXXV) or a composition of (XXXVI) in a method to        diagnose, prophylax, prognose, or treat a cancer of a tissue        listed in Table I; and,    -   (XLII) a method of using a polynucleotide of any (I)-(XXXIV) or        peptide of (XXXV) or a composition of (XXXVI) in a method to        identify or characterize a modulator of a cell expressing        24P4C12.

As used herein, a range is understood to disclose specifically all wholeunit positions thereof.

Typical embodiments of the invention disclosed herein include 24P4C12polynucleotides that encode specific portions of 24P4C12 mRNA sequences(and those which are complementary to such sequences) such as those thatencode the proteins and/or fragments thereof, for example:

(a) 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170,175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 710 or morecontiguous amino acids of 24P4C12 variant 1; the maximal lengthsrelevant for other variants are: variant 3, 710 amino acids; variant 5,710 amino acids, variant 6, 710 amino acids, variant 7, 598 amino acids,variant 8, 722 amino acids, and variant 9, 712 amino acids.

For example, representative embodiments of the invention disclosedherein include: polynucleotides and their encoded peptides themselvesencoding about amino acid 1 to about amino acid 10 of the 24P4C12protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 10 to about amino acid 20 of the 24P4C12 protein shown in FIG. 2 orFIG. 3, polynucleotides encoding about amino acid 20 to about amino acid30 of the 24P4C12 protein shown in FIG. 2 or FIG. 3, polynucleotidesencoding about amino acid 30 to about amino acid 40 of the 24P4C12protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 40 to about amino acid 50 of the 24P4C12 protein shown in FIG. 2 orFIG. 3, polynucleotides encoding about amino acid 50 to about amino acid60 of the 24P4C12 protein shown in FIG. 2 or FIG. 3, polynucleotidesencoding about amino acid 60 to about amino acid 70 of the 24P4C12protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 70 to about amino acid 80 of the 24P4C12 protein shown in FIG. 2 orFIG. 3, polynucleotides encoding about amino acid 80 to about amino acid90 of the 24P4C12 protein shown in FIG. 2 or FIG. 3, polynucleotidesencoding about amino acid 90 to about amino acid 100 of the 24P4C12protein shown in FIG. 2 or FIG. 3, in increments of about 10 aminoacids, ending at the carboxyl terminal amino acid set forth in FIG. 2 orFIG. 3. Accordingly, polynucleotides encoding portions of the amino acidsequence (of about 10 amino acids), of amino acids, 100 through thecarboxyl terminal amino acid of the 24P4C12 protein are embodiments ofthe invention. Wherein it is understood that each particular amino acidposition discloses that position plus or minus five amino acid residues.

Polynucleotides encoding relatively long portions of a 24P4C12 proteinare also within the scope of the invention. For example, polynucleotidesencoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about aminoacid 20, (or 30, or 40 or 50 etc.) of the 24P4C12 protein “or variant”shown in FIG. 2 or FIG. 3 can be generated by a variety of techniqueswell known in the art. These polynucleotide fragments can include anyportion of the 24P4C12 sequence as shown in FIG. 2.

Additional illustrative embodiments of the invention disclosed hereininclude 24P4C12 polynucleotide fragments encoding one or more of thebiological motifs contained within a 24P4C12 protein “or variant”sequence, including one or more of the motif-bearing subsequences of a24P4C12 protein “or variant” set forth in Tables VIII-XXI and XXII-XLIX.In another embodiment, typical polynucleotide fragments of the inventionencode one or more of the regions of 24P4C12 protein or variant thatexhibit homology to a known molecule. In another embodiment of theinvention, typical polynucleotide fragments can encode one or more ofthe 24P4C12 protein or variant N-glycosylation sites, cAMP andcGMP-dependent protein kinase phosphorylation sites, casein kinase IIphosphorylation sites or N-myristoylation site and amidation sites.

Note that to determine the starting position of any peptide set forth inTables VIII-XXI and Tables XXII to XLIX (collectively HLA PeptideTables) respective to its parental protein, e.g., variant 1, variant 2,etc., reference is made to three factors: the particular variant, thelength of the peptide in an HLA Peptide Table, and the Search Peptideslisted in Table LVII. Generally, a unique Search Peptide is used toobtain HLA peptides for a particular variant. The position of eachSearch Peptide relative to its respective parent molecule is listed inTable VII. Accordingly, if a Search Peptide begins at position “X”, onemust add the value “X minus 1” to each position in Tables VIII-XXI andTables XXII-IL to obtain the actual position of the HLA peptides intheir parental molecule. For example if a particular Search Peptidebegins at position 150 of its parental molecule, one must add 150-1,i.e., 149 to each HLA peptide amino acid position to calculate theposition of that amino acid in the parent molecule.

II.A.) Uses of 24P4C12 Polynucleotides

II.A.1.) Monitoring of Genetic Abnormalities

The polynucleotides of the preceding paragraphs have a number ofdifferent specific uses. The human 24P4C12 gene maps to the chromosomallocation set forth in the Example entitled “Chromosomal Mapping of24P4C12.” For example, because the 24P4C12 gene maps to this chromosome,polynucleotides that encode different regions of the 24P4C12 proteinsare used to characterize cytogenetic abnormalities of this chromosomallocale, such as abnormalities that are identified as being associatedwith various cancers. In certain genes, a variety of chromosomalabnormalities including rearrangements have been identified as frequentcytogenetic abnormalities in a number of different cancers (see e.g.Krajinovic et al., Mutat. Res. 382(3-4): 81-83 (1998); Johansson et al.,Blood 86(10): 3905-3914 (1995) and Finger etaaL, P.N.A.S. 85(23):9158-9162 (1988)). Thus, polynucleotides encoding specific regions ofthe 24P4C12 proteins provide new tools that can be used to delineate,with greater precision than previously possible, cytogeneticabnormalities in the chromosomal region that encodes 24P4C12 that maycontribute to the malignant phenotype. In this context, thesepolynucleotides satisfy a need in the art for expanding the sensitivityof chromosomal screening in order to identify more subtle and lesscommon chromosomal abnormalities (see e.g. Evans et al., Am. J. Obstet.Gynecol 171(4): 1055-1057 (1994)).

Furthermore, as 24P4C12 was shown to be highly expressed in bladder andother cancers, 24P4C12 polynucleotides are used in methods assessing thestatus of 24P4C12 gene products in normal versus cancerous tissues.Typically, polynucleotides that encode specific regions of the 24P4C12proteins are used to assess the presence of perturbations (such asdeletions, insertions, point mutations, or alterations resulting in aloss of an antigen etc.) in specific regions of the 24P4C12 gene, suchas regions containing one or more motifs. Exemplary assays include bothRT-PCR assays as well as single-strand conformation polymorphism (SSCP)analysis (see, e.g., Marrogi et al., J. Cutan. Pathol. 26(8): 369-378(1999), both of which utilize polynucleotides encoding specific regionsof a protein to examine these regions within the protein.

II.A.2.) Antisense Embodiments

Other specifically contemplated nucleic acid related embodiments of theinvention disclosed herein are genomic DNA, cDNAs, ribozymes, andantisense molecules, as well as nucleic acid molecules based on analternative backbone, or including alternative bases, whether derivedfrom natural sources or synthesized, and include molecules capable ofinhibiting the RNA or protein expression of 24P4C12. For example,antisense molecules can be RNAs or other molecules, including peptidenucleic acids (PNAs) or non-nucleic acid molecules such asphosphorothioate derivatives that specifically bind DNA or RNA in a basepair-dependent manner. A skilled artisan can readily obtain theseclasses of nucleic acid molecules using the 24P4C12 polynucleotides andpolynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenousoligonucleotides that bind to a target polynucleotide located within thecells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.,24P4C12. See for example, Jack Cohen, Oligodeoxynucleotides, AntisenseInhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5(1988). The 24P4C12 antisense oligonucleotides of the present inventioninclude derivatives such as S-oligonucleotides (phosphorothioatederivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhancedcancer cell growth inhibitory action. S-oligos (nucleosidephosphorothioates) are isoelectronic analogs of an oligonucleotide(O-oligo) in which a nonbridging oxygen atom of the phosphate group isreplaced by a sulfur atom. The S-oligos of the present invention can beprepared by treatment of the corresponding O-oligos with3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transferreagent. See, e.g., Iyer, R. P. et al., J. Org. Chem. 55:4693-4698(1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990).Additional 24P4C12 antisense oligonucleotides of the present inventioninclude morpholino antisense oligonucleotides known in the art (see,e.g., Partridge et al., 1996, Antisense & Nucleic Acid Drug Development6: 169-175).

The 24P4C12 antisense oligonucleotides of the present inventiontypically can be RNA or DNA that is complementary to and stablyhybridizes with the first 100 5′ codons or last 100 3′ codons of a24P4C12 genomic sequence or the corresponding mRNA. Absolutecomplementarity is not required, although high degrees ofcomplementarity are preferred. Use of an oligonucleotide complementaryto this region allows for the selective hybridization to 24P4C12 mRNAand not to mRNA specifying other regulatory subunits of protein kinase.In one embodiment, 24P4C12 antisense oligonucleotides of the presentinvention are 15 to 30-mer fragments of the antisense DNA molecule thathave a sequence that hybridizes to 24P4C12 mRNA. Optionally, 24P4C12antisense oligonucleotide is a 30-mer oligonucleotide that iscomplementary to a region in the first 10 5′ codons or last 10 3′ codonsof 24P4C12. Alternatively, the antisense molecules are modified toemploy ribozymes in the inhibition of 24P4C12 expression, see, e.g., L.A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).

II.A.3.) Primers and Primer Pairs

Further specific embodiments of these nucleotides of the inventioninclude primers and primer pairs, which allow the specific amplificationof polynucleotides of the invention or of any specific parts thereof,and probes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes can be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers are used todetect the presence of a 24P4C12 polynucleotide in a sample and as ameans for detecting a cell expressing a 24P4C12 protein.

Examples of such probes include polypeptides comprising all or part ofthe human 24P4C12 cDNA sequence shown in FIG. 2. Examples of primerpairs capable of specifically amplifying 24P4C12 mRNAs are alsodescribed in the Examples. As will be understood by the skilled artisan,a great many different primers and probes can be prepared based on thesequences provided herein and used effectively to amplify and/or detecta 24P4C12 mRNA.

The 24P4C12 polynucleotides of the invention are useful for a variety ofpurposes, including but not limited to their use as probes and primersfor the amplification and/or detection of the 24P4C12 gene(s), mRNA(s),or fragments thereof; as reagents for the diagnosis and/or prognosis ofprostate cancer and other cancers; as coding sequences capable ofdirecting the expression of 24P4C12 polypeptides; as tools formodulating or inhibiting the expression of the 24P4C12 gene(s) and/ortranslation of the 24P4C12 transcript(s); and as therapeutic agents.

The present invention includes the use of any probe as described hereinto identify and isolate a 24P4C12 or 24P4C12 related nucleic acidsequence from a naturally occurring source, such as humans or othermammals, as well as the isolated nucleic acid sequence per se, whichwould comprise all or most of the sequences found in the probe used.

II.A.4.) Isolation of 24P4C12-Encoding Nucleic Acid Molecules

The 24P4C12 cDNA sequences described herein enable the isolation ofother polynucleotides encoding 24P4C12 gene product(s), as well as theisolation of polynucleotides encoding 24P4C12 gene product homologs,alternatively spliced isoforms, allelic variants, and mutant forms of a24P4C12 gene product as well as polynucleotides that encode analogs of24P4C12-related proteins. Various molecular cloning methods that can beemployed to isolate full length cDNAs encoding a 24P4C12 gene are wellknown (see, for example, Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989;Current Protocols in Molecular Biology. Ausubel et al., Eds., Wiley andSons, 1995). For example, lambda phage cloning methodologies can beconveniently employed, using commercially available cloning systems(e.g., Lambda ZAP Express, Stratagene). Phage clones containing 24P4C12gene cDNAs can be identified by probing with a labeled 24P4C12 cDNA or afragment thereof. For example, in one embodiment, a 24P4C12 cDNA (e.g.,FIG. 2) or a portion thereof can be synthesized and used as a probe toretrieve overlapping and full-length cDNAs corresponding to a 24P4C12gene. A 24P4C12 gene itself can be isolated by screening genomic DNAlibraries, bacterial artificial chromosome libraries (BACs), yeastartificial chromosome libraries (YACs), and the like, with 24P4C12 DNAprobes or primers.

II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containinga 24P4C12 polynucleotide, a fragment, analog or homologue thereof,including but not limited to phages, plasmids, phagemids, cosmids, YACs,BACs, as well as various viral and non-viral vectors well known in theart, and cells transformed or transfected with such recombinant DNA orRNA molecules. Methods for generating such molecules are well known(see, for example, Sambrook et al., 1989, supra).

The invention further provides a host-vector system comprising arecombinant DNA molecule containing a 24P4C12 polynucleotide, fragment,analog or homologue thereof within a suitable prokaryotic or eukaryotichost cell. Examples of suitable eukaryotic host cells include a yeastcell, a plant cell, or an animal cell, such as a mammalian cell or aninsect cell (e.g., a baculovirus-infectible cell such as an Sf9 orHighFive cell). Examples of suitable mammalian cells include variousprostate cancer cell lines such as DU145 and TsuPr1, other transfectableor transducible prostate cancer cell lines, primary cells (PrEC), aswell as a number of mammalian cells routinely used for the expression ofrecombinant proteins (e.g., COS, CHO, 293, 293T cells). Moreparticularly, a polynucleotide comprising the coding sequence of 24P4C12or a fragment, analog or homolog thereof can be used to generate 24P4C12proteins or fragments thereof using any number of host-vector systemsroutinely used and widely known in the art.

A wide range of host-vector systems suitable for the expression of24P4C12 proteins or fragments thereof are available, see for example,Sambrook et al., 1989, supra; Current Protocols in Molecular Biology,1995, supra). Preferred vectors for mammalian expression include but arenot limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviralvector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using theseexpression vectors, 24P4C12 can be expressed in several prostate cancerand non-prostate cell lines, including for example 293, 293T, rat-1, NIH3T3 and TsuPr1. The host-vector systems of the invention are useful forthe production of a 24P4C12 protein or fragment thereof. Suchhost-vector systems can be employed to study the functional propertiesof 24P4C12 and 24P4C12 mutations or analogs.

Recombinant human 24P4C12 protein or an analog or homolog or fragmentthereof can be produced by mammalian cells transfected with a constructencoding a 24P4C12-related nucleotide. For example, 293T cells can betransfected with an expression plasmid encoding 24P4C12 or fragment,analog or homolog thereof, a 24P4C12-related protein is expressed in the293T cells, and the recombinant 24P4C12 protein is isolated usingstandard purification methods (e.g., affinity purification usinganti-24P4C12 antibodies). In another embodiment, a 24P4C12 codingsequence is subcloned into the retroviral vector pSRαMSVtkneo and usedto infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293 andrat-1 in order to establish 24P4C12 expressing cell lines. Various otherexpression systems well known in the art can also be employed.Expression constructs encoding a leader peptide joined in frame to a24P4C12 coding sequence can be used for the generation of a secretedform of recombinant 24P4C12 protein.

As discussed herein, redundancy in the genetic code permits variation in24P4C12 gene sequences. In particular, it is known in the art thatspecific host species often have specific codon preferences, and thusone can adapt the disclosed sequence as preferred for a desired host.For example, preferred analog codon sequences typically have rare codons(i.e., codons having a usage frequency of less than about 20% in knownsequences of the desired host) replaced with higher frequency codons.Codon preferences for a specific species are calculated, for example, byutilizing codon usage tables available on the INTERNET such as at URLdna.affrc.go.jp/˜nakamura/codon.html.

Additional sequence modifications are known to enhance proteinexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon/intron splice sitesignals, transposon-like repeats, and/or other such well-characterizedsequences that are deleterious to gene expression. The GC content of thesequence is adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Wherepossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures. Other useful modifications include the addition of atranslational initiation consensus sequence at the start of the openreading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080(1989). Skilled artisans understand that the general rule thateukaryotic ribosomes initiate translation exclusively at the 5′ proximalAUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).

III.) 24P4C12-RELATED PROTEINS

Another aspect of the present invention provides 24P4C12-relatedproteins. Specific embodiments of 24P4C12 proteins comprise apolypeptide having all or part of the amino acid sequence of human24P4C12 as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of24P4C12 proteins comprise variant, homolog or analog polypeptides thathave alterations in the amino acid sequence of 24P4C12 shown in FIG. 2or FIG. 3.

Embodiments of a 24P4C12 polypeptide include: a 24P4C12 polypeptidehaving a sequence shown in FIG. 2, a peptide sequence of a 24P4C12 asshown in FIG. 2 wherein T is U; at least 10 contiguous nucleotides of apolypeptide having the sequence as shown in FIG. 2; or, at least 10contiguous peptides of a polypeptide having the sequence as shown inFIG. 2 where T is U. For example, embodiments of 24P4C12 peptidescomprise, without limitation:

-   -   (I) a protein comprising, consisting essentially of, or        consisting of an amino acid sequence as shown in FIG. 2A-I or        FIGS. 3A-G;    -   (II) a 24P4C12-related protein that is at least 90, 91, 92, 93,        94, 95, 96, 97, 98, 99 or 100% homologous to an entire amino        acid sequence shown in FIGS. 2A-I;    -   (III) a 24P4C12-related protein that is at least 90, 91, 92, 93,        94, 95, 96, 97, 98, 99 or 100% identical to an entire amino acid        sequence shown in FIGS. 2A-I or 3A-G;    -   (IV) a protein that comprises at least one peptide set forth in        Tables VIII to XLIX, optionally with a proviso that it is not an        entire protein of FIG. 2;    -   (V) a protein that comprises at least one peptide set forth in        Tables VIII-XXI, collectively, which peptide is also set forth        in Tables XXII to XLIX, collectively, optionally with a proviso        that it is not an entire protein of FIG. 2;    -   (VI) a protein that comprises at least two peptides selected        from the peptides set forth in Tables VIII-XLIX, optionally with        a proviso that it is not an entire protein of FIG. 2;    -   (VII) a protein that comprises at least two peptides selected        from the peptides set forth in Tables VIII to XLIX collectively,        with a proviso that the protein is not a contiguous sequence        from an amino acid sequence of FIG. 2;    -   (VIII) a protein that comprises at least one peptide selected        from the peptides set forth in Tables VIII-XXI; and at least one        peptide selected from the peptides set forth in Tables XXII to        XLIX, with a proviso that the protein is not a contiguous        sequence from an amino acid sequence of FIG. 2;    -   (IX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10,11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,        29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIGS. 3A,        3B, 3C, 3D, 3E, 3F, or 3G in any whole number increment up to        710, 710, 710, 710, 598, 722, or 712 respectively that includes        at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,        33, 34, 35 amino acid position(s) having a value greater than        0.5 in the Hydrophilicity profile of FIG. 5;    -   (X) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,        29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIGS. 3A,        3B, 3C, 3D, 3E, 3F, or 3G in any whole number increment up to        710, 710, 710, 710, 598, 722, or 712 respectively, that includes        at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,        33, 34, 35 amino acid position(s) having a value less than 0.5        in the Hydropathicity profile of FIG. 6;    -   (XI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIGS.        3A, 3B, 3C, 3D, 3E, 3F, or 3G in any whole number increment up        to 710, 710, 710, 710, 598, 722, or 712 respectively, that        includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35 amino acid position(s) having a value greater        than 0.5 in the Percent Accessible Residues profile of FIG. 7;    -   (XII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIGS.        3A, 3B, 3C, 3D, 3E, 3F, or 3G in any whole number increment up        to 710, 710, 710, 710, 598, 722, or 712 respectively, that        includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35-amino acid position(s) having a value greater        than 0.5 in the Average Flexibility profile of FIG. 8;    -   (XIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIGS.        3A, 3B, 3C, 3D, 3E, 3F, or 3G in any whole number increment up        to 710, 710, 710, 710, 598, 722, or 712 respectively, that        includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35 amino acid position(s) having a value greater        than 0.5 in the Beta-turn profile of FIG. 9;    -   (XIV) a peptide that occurs at least twice in Tables VIII-XXI        and XXII to XLIX, collectively;    -   (XV) a peptide that occurs at least three times in Tables        VIII-XXI and XXII to XLIX, collectively;    -   (XVI) a peptide that occurs at least four times in Tables        VIII-XXI and XXII to XLIX, collectively;    -   (XVII) a peptide that occurs at least five times in Tables        VIII-XXI and XXII to XLIX, collectively;    -   (XVIII) a peptide that occurs at least once in Tables VIII-XXI,        and at least once in tables XXII to XLIX;    -   (XIX) a peptide that occurs at least once in Tables VIII-XXI,        and at least twice in tables XXII to XLIX;    -   (XX) a peptide that occurs at least twice in Tables VIII-XXI,        and at least once in tables XXII to XLIX;    -   (XXI) a peptide that occurs at least twice in Tables VIII-XXI,        and at least twice in tables XXII to XLIX;    -   (XXII) a peptide which comprises one two, three, four, or five        of the following characteristics, or an oligonucleotide encoding        such peptide:        -   i) a region of at least 5 amino acids of a particular            peptide of FIG. 3, in any whole number increment up to the            full length of that protein in FIG. 3, that includes an            amino acid position having a value equal to or greater than            0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in            the Hydrophilicity profile of FIG. 5;        -   ii) a region of at least 5 amino acids of a particular            peptide of FIG. 3, in any whole number increment up to the            full length of that protein in FIG. 3, that includes an            amino acid position having a value equal to or less than            0.5, 0.4, 0.3, 0.2, 0.1, or having a value equal to 0.0, in            the Hydropathicity profile of FIG. 6;        -   iii) a region of at least 5 amino acids of a particular            peptide of FIG. 3, in any whole number increment up to the            full length of that protein in FIG. 3, that includes an            amino acid position having a value equal to or greater than            0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in            the Percent Accessible Residues profile of FIG. 7;        -   iv) a region of at least 5 amino acids of a particular            peptide of FIG. 3, in any whole number increment up to the            full length of that protein in FIG. 3, that includes an            amino acid position having a value equal to or greater than            0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in            the Average Flexibility profile of FIGS. 8; or,        -   v) a region of at least 5 amino acids of a particular            peptide of FIG. 3, in any whole number increment up to the            full length of that protein in FIG. 3, that includes an            amino acid position having a value equal to or greater than            0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in            the Beta-turn profile of FIG. 9;    -   (XXIII) a composition comprising a peptide of (I)-(XXII) or an        antibody or binding region thereof together with a        pharmaceutical excipient and/or in a human unit dose form.    -   (XXIV) a method of using a peptide of (I)-(XXII), or an antibody        or binding region thereof or a composition of (XXIII) in a        method to modulate a cell expressing 24P4C12,    -   (XXV) a method of using a peptide of (I)-(XXII) or an antibody        or binding region thereof or a composition of (XXIII) in a        method to diagnose, prophylax, prognose, or treat an individual        who bears a cell expressing 24P4C12    -   (XXVI) a method of using a peptide of (I)-(XXII) or an antibody        or binding region thereof or a composition (XXIII) in a method        to diagnose, prophylax, prognose, or treat an individual who        bears a cell expressing 24P4C12, said cell from a cancer of a        tissue listed in Table I;    -   (XXVII) a method of using a peptide of (I)-(XXII) or an antibody        or binding region thereof or a composition of (XXIII) in a        method to diagnose, prophylax, prognose, or treat a cancer;    -   (XXVIII) a method of using a peptide of (I)-(XXII) or an        antibody or binding region thereof or a composition of (XXIII)        in a method to diagnose, prophylax, prognose, or treat a cancer        of a tissue listed in Table I; and,    -   (XXIX) a method of using a a peptide of (I)-(XXII) or an        antibody or binding region thereof or a composition (XXIII) in a        method to identify or characterize a modulator of a cell        expressing 24P4C12.

As used herein, a range is understood to specifically disclose all wholeunit positions thereof.

Typical embodiments of the invention disclosed herein include 24P4C12polynucleotides that encode specific portions of 24P4C12 mRNA sequences(and those which are complementary to such sequences) such as those thatencode the proteins and/or fragments thereof, for example:

(a) 4, 5, 6, 7, 8, 9, 10, 11,12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170,175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 710 or morecontiguous amino acids of 24P4C12 variant 1; the maximal lengthsrelevant for other variants are: variant 3, 710 amino acids; variant 5,710 amino acids, variant 6, 710, variant 7, 598 amino acids, variant 8,722 amino acids, and variant 9, 712 amino acids.

In general, naturally occurring allelic variants of human 24P4C12 sharea high degree of structural identity and homology (e.g., 90% or morehomology). Typically, allelic variants of a 24P4C12 protein containconservative amino acid substitutions within the 24P4C12 sequencesdescribed herein or contain a substitution of an amino acid from acorresponding position in a homologue of 24P4C12. One class of 24P4C12allelic variants are proteins that share a high degree of homology withat least a small region of a particular 24P4C12 amino acid sequence, butfurther contain a radical departure from the sequence, such as anon-conservative substitution, truncation, insertion or frame shift. Incomparisons of protein sequences, the terms, similarity, identity, andhomology each have a distinct meaning as appreciated in the field ofgenetics. Moreover, orthology and paralogy can be important conceptsdescribing the relationship of members of a given protein family in oneorganism to the members of the same family in other organisms.

Amino acid abbreviations are provided in Table II. Conservative aminoacid substitutions can frequently be made in a protein without alteringeither the conformation or the function of the protein. Proteins of theinvention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15conservative substitutions. Such changes include substituting any ofisoleucine (I), valine (V), and leucine (L) for any other of thesehydrophobic amino acids; aspartic acid (D) for glutamic acid (E) andvice versa; glutamine (Q) for asparagine (N) and vice versa; and serine(S) for threonine (T) and vice versa. Other substitutions can also beconsidered conservative, depending on the environment of the particularamino acid and its role in the three-dimensional structure of theprotein. For example, glycine (G) and alanine (A) can frequently beinterchangeable, as can alanine (A) and valine (V). Methionine (M),which is relatively hydrophobic, can frequently be interchanged withleucine and isoleucine, and sometimes with valine. Lysine (K) andarginine (R) are frequently interchangeable in locations in which thesignificant feature of the amino acid residue is its charge and thediffering pK's of these two amino acid residues are not significant.Still other changes can be considered “conservative” in particularenvironments (see, e.g. Table III herein; pages 13-15 “Biochemistry”2^(nd) ED. Lubert Stryer ed (Stanford University); Henikoff et al., PNAS1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19;270(20):11882-6).

Embodiments of the invention disclosed herein include a wide variety ofart-accepted variants or analogs of 24P4C12 proteins such aspolypeptides having amino acid insertions, deletions and substitutions.24P4C12 variants can be made using methods known in the art such assite-directed mutagenesis, alanine scanning, and PCR mutagenesis.Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331(1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassettemutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selectionmutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415(1986)) or other known techniques can be performed on the cloned DNA toproduce the 24P4C12 variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence that is involved in aspecific biological activity such as a protein-protein interaction.Among the preferred scanning amino acids are relatively small, neutralamino acids. Such amino acids include alanine, glycine, serine, andcysteine. Alanine is typically a preferred scanning amino acid amongthis group because it eliminates the side-chain beyond the beta-carbonand is less likely to alter the main-chain conformation of the variant.Alanine is also typically preferred because it is the most common aminoacid. Further, it is frequently found in both buried and exposedpositions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia,J. Mol. Biol., 150:1 (1976)). If alanine substitution does not yieldadequate amounts of variant, an isosteric amino acid can be used.

As defined herein, 24P4C12 variants, analogs or homologs, have thedistinguishing attribute of having at least one epitope that is “crossreactive” with a 24P4C12 protein having an amino acid sequence of FIG.3. As used in this sentence, “cross reactive” means that an antibody orT cell that specifically binds to a 24P4C12 variant also specificallybinds to a 24P4C12 protein having an amino acid sequence set forth inFIG. 3. A polypeptide ceases to be a variant of a protein shown in FIG.3, when it no longer contains any epitope capable of being recognized byan antibody or T cell that specifically binds to the starting 24P4C12protein. Those skilled in the art understand that antibodies thatrecognize proteins bind to epitopes of varying size, and a grouping ofthe order of about four or five amino acids, contiguous or not, isregarded as a typical number of amino acids in a minimal epitope. See,e.g., Nair et al., J. Immunol. 2000 165(12): 6949-6955; Hebbes et al.,Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol (1985)135(4):2598-608.

Other classes of 24P4C12-related protein variants share 70%, 75%, 80%,85% or 90% or more similarity with an amino acid sequence of FIG. 3, ora fragment thereof. Another specific class of 24P4C12 protein variantsor analogs comprises one or more of the 24P4C12 biological motifsdescribed herein or presently known in the art. Thus, encompassed by thepresent invention are analogs of 24P4C12 fragments (nucleic or aminoacid) that have altered functional (e.g. immunogenic) propertiesrelative to the starting fragment. It is to be appreciated that motifsnow or which become part of the art are to be applied to the nucleic oramino acid sequences of FIG. 2 or FIG. 3.

As discussed herein, embodiments of the claimed invention includepolypeptides containing less than the full amino acid sequence of a24P4C12 protein shown in FIG. 2 or FIG. 3. For example, representativeembodiments of the invention comprise peptides/proteins having any 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of a24P4C12 protein shown in FIG. 2 or FIG. 3.

Moreover, representative embodiments of the invention disclosed hereininclude polypeptides consisting of about amino acid 1 to about aminoacid 10 of a 24P4C12 protein shown in FIG. 2 or FIG. 3, polypeptidesconsisting of about amino acid 10 to about amino acid 20 of a 24P4C12protein shown in FIG. 2 or FIG. 3, polypeptides consisting of aboutamino acid 20 to about amino acid 30 of a 24P4C12 protein shown in FIG.2 or FIG. 3, polypeptides consisting of about amino acid 30 to aboutamino acid 40 of a 24P4C12 protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 40 to about amino acid 50 ofa 24P4C12 protein shown in FIG. 2 or FIG. 3, polypeptides consisting ofabout amino acid 50 to about amino acid 60 of a 24P4C12 protein shown inFIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60 toabout amino acid 70 of a 24P4C12 protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 70 to about amino acid 80 ofa 24P4C12 protein shown in FIG. 2 or FIG. 3, polypeptides consisting ofabout amino acid 80 to about amino acid 90 of a 24P4C12 protein shown inFIG. 2 or FIG. 3, polypeptides consisting of about amino acid 90 toabout amino acid 100 of a 24P4C12 protein shown in FIG. 2 or FIG. 3,etc. throughout the entirety of a 24P4C12 amino acid sequence. Moreover,polypeptides consisting of about amino acid 1 (or 20 or 30 or 40 etc.)to about amino acid 20, (or 130, or 140 or 150 etc.) of a 24P4C12protein shown in FIG. 2 or FIG. 3 are embodiments of the invention. Itis to be appreciated that the starting and stopping positions in thisparagraph refer to the specified position as well as that position plusor minus 5 residues.

24P4C12-related proteins are generated using standard peptide synthesistechnology or using chemical cleavage methods well known in the art.Alternatively, recombinant methods can be used to generate nucleic acidmolecules that encode a 24P4C12-related protein. In one embodiment,nucleic acid molecules provide a means to generate defined fragments ofa 24P4C12 protein (or variants, homologs or analogs thereof).

III.A.) Motif-Bearing Protein Embodiments

Additional illustrative embodiments of the invention disclosed hereininclude 24P4C12 polypeptides comprising the amino acid residues of oneor more of the biological motifs contained within a 24P4C12 polypeptidesequence set forth in FIG. 2 or FIG. 3. Various motifs are known in theart, and a protein can be evaluated for the presence of such motifs by anumber of publicly available Internet sites (see, e.g., URL addresses:pfam.wustl.edu/;searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html;psort.ims.u-tokyo.ac.jp/; cbs.dtu.dk/; ebi.ac.uk/interpro/scan.html;expasy.ch/tools/scnpsit1.html; Epimatrix™ and Epimer™, Brown University,brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS,bimas.dcrt.nih.gov/.).

Motif bearing subsequences of all 24P4C12 variant proteins are set forthand identified in Tables VIII-XXI and XXII-XLIX.

Table V sets forth several frequently occurring motifs based on pfamsearches (see URL address pfam.wustl.edu/). The columns of Table V list(1) motif name abbreviation, (2) percent identity found amongst thedifferent member of the motif family, (3) motif name or description and(4) most common function; location information is included if the motifis relevant for location.

Polypeptides comprising one or more of the 24P4C12 motifs discussedabove are useful in elucidating the specific characteristics of amalignant phenotype in view of the observation that the 24P4C12 motifsdiscussed above are associated with growth dysregulation and because24P4C12 is overexpressed in certain cancers (See, e.g., Table I). Caseinkinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C,for example, are enzymes known to be associated with the development ofthe malignant phenotype (see e.g. Chen et al., Lab Invest., 78(2):165-174 (1998); Gaiddon et al., Endocrinology 136(10): 4331-4338 (1995);Hall et al., Nucleic Acids Research 24(6): 1119-1126 (1996); Peterzielet af., Oncogene 18(46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 5(2):305-309 (1998)). Moreover, both glycosylation and myristoylation areprotein modifications also associated with cancer and cancer progression(see e.g. Dennis et al., Biochem. Biophys. Acta 1473(1):21-34 (1999);Raju et al., Exp. Cell Res. 235(1): 145-154 (1997)). Amidation isanother protein modification also associated with cancer and cancerprogression (see e.g. Treston et al., J. Natl. Cancer Inst. Monogr.(13): 169-175 (1992)).

In another embodiment, proteins of the invention comprise one or more ofthe immunoreactive epitopes identified in accordance with art-acceptedmethods, such as the peptides set forth in Tables VIII-XXI andXXII-XLIX. CTL epitopes can be determined using specific algorithms toidentify peptides within a 24P4C12 protein that are capable of optimallybinding to specified HLA alleles (e.g., Table IV; Epimatrix™ andEpimer™, Brown University, URLbrown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS, URLbimas.dcrt.nih.gov/.) Moreover, processes for identifying peptides thathave sufficient binding affinity for HLA molecules and which arecorrelated with being immunogenic epitopes, are well known in the art,and are carried out without undue experimentation. In addition,processes for identifying peptides that are immunogenic epitopes, arewell known in the art, and are carried out without undue experimentationeither in vitro or in vivo.

Also known in the art are principles for creating analogs of suchepitopes in order to modulate immunogenicity. For example, one beginswith an epitope that bears a CTL or HTL motif (see, e.g., the HLA ClassI and HLA Class II motifs/supermotifs of Table IV). The epitope isanaloged by substituting out an amino acid at one of the specifiedpositions, and replacing it with another amino acid specified for thatposition. For example, on the basis of residues defined in Table IV, onecan substitute out a deleterious residue in favor of any other residue,such as a preferred residue; substitute a less-preferred residue with apreferred residue; or substitute an originally-occurring preferredresidue with another preferred residue. Substitutions can occur atprimary anchor positions or at other positions in a peptide; see, e.g.,Table IV.

A variety of references reflect the art regarding the identification andgeneration of epitopes in a protein of interest as well as analogsthereof. See, for example, WO 97/33602 to Chesnut et al.; Sette,Immunogenetics 1999 50(3-4): 201-212; Sette et al., J. Immunol. 2001166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondoet al., Immunogenetics 1997 45(4): 249-258; Sidney et al, J. Immunol.1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt etal., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7(1992); Parker et al., J. Immunol. 152:163-75 (1994)); Kast et al., 1994152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3):266-278; Alexander et al., J. Immunol, 2000 164(3); 164(3): 1625-1633;Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al., J.Immunol. 1991 147(8): 2663-2669; Alexander et al., Immunity 1994 1(9):751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92.

Related embodiments of the invention include polypeptides comprisingcombinations of the different motifs set forth in Table VI, and/or, oneor more of the predicted CTL epitopes of Tables VIII-XXI and XXII-XLIX,and/or, one or more of the predicted HTL epitopes of Tables XLVI-XLIX,and/or, one or more of the T cell binding motifs known in the art.Preferred embodiments contain no insertions, deletions or substitutionseither within the motifs or within the intervening sequences of thepolypeptides. In addition, embodiments which include a number of eitherN-terminal and/or C-terminal amino acid residues on either side of thesemotifs may be desirable (to, for example, include a greater portion ofthe polypeptide architecture in which the motif is located). Typically,the number of N-terminal and/or C-terminal amino acid residues on eitherside of a motif is between about 1 to about 100 amino acid residues,preferably 5 to about 50 amino acid residues.

24P4C12-related proteins are embodied in many forms, preferably inisolated form. A purified 24P4C12 protein molecule will be substantiallyfree of other proteins or molecules that impair the binding of 24P4C12to antibody, T cell or other ligand. The nature and degree of isolationand purification will depend on the intended use. Embodiments of a24P4C12-related proteins include purified 24P4C12-related proteins andfunctional, soluble 24P4C12-related proteins. In one embodiment, afunctional, soluble 24P4C12 protein or fragment thereof retains theability to be bound by antibody, T cell or other ligand.

The invention also provides 24P4C12 proteins comprising biologicallyactive fragments of a 24P4C12 amino acid sequence shown in FIG. 2 orFIG. 3. Such proteins exhibit properties of the starting 24P4C12protein, such as the ability to elicit the generation of antibodies thatspecifically bind an epitope associated with the starting 24P4C12protein; to be bound by such antibodies; to elicit the activation of HTLor CTL; and/or, to be recognized by HTL or CTL that also specificallybind to the starting protein.

24P4C12-related polypeptides that contain particularly interestingstructures can be predicted and/or identified using various analyticaltechniques well known in the art, including, for example, the methods ofChou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis, or based on immunogenicity. Fragments thatcontain such structures are particularly useful in generatingsubunit-specific anti-24P4C12 antibodies or T cells or in identifyingcellular factors that bind to 24P4C12. For example, hydrophilicityprofiles can be generated, and immunogenic peptide fragments identified,using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl.Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can begenerated, and immunogenic peptide fragments identified, using themethod of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol.157:105-132. Percent (%)Accessible Residues profiles can be generated,and immunogenic peptide fragments identified, using the method of JaninJ., 1979, Nature 277:491-492. Average Flexibility profiles can begenerated, and immunogenic peptide fragments identified, using themethod of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. ProteinRes. 32:242-255. Beta-turn profiles can be generated, and immunogenicpeptide fragments identified, using the method of Deleage, G., Roux B.,1987, Protein Engineering 1:289-294.

CTL epitopes can be determined using specific algorithms to identifypeptides within a 24P4C12 protein that are capable of optimally bindingto specified HLA alleles (e.g., by using the SYFPEITHI site at WorldWide Web URL syfpeithi.bmi-heidelberg.com/; the listings in TableIV(A)-(E); Epimatrix™ and Epimer™, Brown University, URL(brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and BIMAS, URLbimas.dcrt.nih.gov/). Illustrating this, peptide epitopes from 24P4C12that are presented in the context of human MHC Class I molecules, e.g.,HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (see, e.g., TablesVIII-XXI, XXII-XLIX). Specifically, the complete amino acid sequence ofthe 24P4C12 protein and relevant portions of other variants, i.e., forHLA Class I predictions 9 flanking residues on either side of a pointmutation or exon junction, and for HLA Class II predictions 14 flankingresidues on either side of a point mutation or exon junctioncorresponding to that variant, were entered into the HLA Peptide MotifSearch algorithm found in the Bioinformatics and Molecular AnalysisSection (BIMAS) web site listed above; in addition to the siteSYFPEITHI, at URL syfpeithi.bmi-heidelberg.com/.

The HLA peptide motif search algorithm was developed by Dr. Ken Parkerbased on binding of specific peptide sequences in the groove of HLAClass I molecules, in particular HLA-A2 (see, e.g., Falk et al., Nature351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker etal., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol.152:163-75 (1994)). This algorithm allows location and ranking of 8-mer,9-mer, and 10-mer peptides from a complete protein sequence forpredicted binding to HLA-A2 as well as numerous other HLA Class Imolecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers.For example, for Class I HLA-A2, the epitopes preferably contain aleucine (L) or methionine (M) at position 2 and a valine (V) or leucine(L) at the C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7(1992)). Selected results of 24P4C12 predicted binding peptides areshown in Tables VIII-XXI and XXII-XLIX herein. In Tables VIII-XXI andXXII-XLVII, selected candidates, 9-mers and 10-mers, for each familymember are shown along with their location, the amino acid sequence ofeach specific peptide, and an estimated binding score. In TablesXLVI-XLIX, selected candidates, 15-mers, for each family member areshown along with their location, the amino acid sequence of eachspecific peptide, and an estimated binding score. The binding scorecorresponds to the estimated half time of dissociation of complexescontaining the peptide at 37° C. at pH 6.5. Peptides with the highestbinding score are predicted to be the most tightly bound to HLA Class Ion the cell surface for the greatest period of time and thus representthe best immunogenic targets for T-cell recognition.

Actual binding of peptides to an HLA allele can be evaluated bystabilization of HLA expression on the antigen-processing defective cellline T2 (see, e.g., Xue et al., Prostate 30:73-8 (1997) and Peshwa etal., Prostate 36:129-38 (1998)). Immunogenicity of specific peptides canbe evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes(CTL) in the presence of antigen presenting cells such as dendriticcells.

It is to be appreciated that every epitope predicted by the BIMAS site,Epimer™ and Epimatrix™ sites, or specified by the HLA class I or classII motifs available in the art or which become part of the art such asset forth in Table IV (or determined using World Wide Web site URLsyfpeithi.bmi-heidelberg.com/, or BIMAS, bimas.dcrt.nih.gov/) are to be“applied” to a 24P4C12 protein in accordance with the invention. As usedin this context “applied” means that a 24P4C12 protein is evaluated,e.g., visually or by computer-based patterns finding methods, asappreciated by those of skill in the relevant art. Every subsequence ofa 24P4C12 protein of 8, 9, 10, or 11 amino acid residues that bears anHLA Class I motif, or a subsequence of 9 or more amino acid residuesthat bear an HLA Class II motif are within the scope of the invention.

III.B.) Expression of 24P4C12-Related Proteins

In an embodiment described in the examples that follow, 24P4C12 can beconveniently expressed in cells (such as 293T cells) transfected with acommercially available expression vector such as a CMV-driven expressionvector encoding 24P4C12 with a C-terminal 6×His and MYC tag(pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, NashvilleTenn.). The Tag5 vector provides an IgGK secretion signal that can beused to facilitate the production of a secreted 24P4C12 protein intransfected cells. The secreted HIS-tagged 24P4C12 in the culture mediacan be purified, e.g., using a nickel column using standard techniques.

III.C.) Modifications of 24P4C12-Related Proteins

Modifications of 24P4C12-related proteins such as covalent modificationsare included within the scope of this invention. One type of covalentmodification includes reacting targeted amino acid residues of a 24P4C12polypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues of a24P4C12 protein. Another type of covalent modification of a 24P4C12polypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of a protein of the invention.Another type of covalent modification of 24P4C12 comprises linking a24P4C12 polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The 24P4C12-related proteins of the present invention can also bemodified to form a chimeric molecule comprising 24P4C12 fused toanother, heterologous polypeptide or amino acid sequence. Such achimeric molecule can be synthesized chemically or recombinantly. Achimeric molecule can have a protein of the invention fused to anothertumor-associated antigen or fragment thereof. Alternatively, a proteinin accordance with the invention can comprise a fusion of fragments of a24P4C12 sequence (amino or nucleic acid) such that a molecule is createdthat is not, through its length, directly homologous to the amino ornucleic acid sequences shown in FIG. 2 or FIG. 3. Such a chimericmolecule can comprise multiples of the same subsequence of 24P4C12. Achimeric molecule can comprise a fusion of a 24P4C12-related proteinwith a polyhistidine epitope tag, which provides an epitope to whichimmobilized nickel can selectively bind, with cytokines or with growthfactors. The epitope tag is generally placed at the amino- orcarboxyl-terminus of a 24P4C12 protein. In an alternative embodiment,the chimeric molecule can comprise a fusion of a 24P4C12-related proteinwith an immunoglobulin or a particular region of an immunoglobulin. Fora bivalent form of the chimeric molecule (also referred to as an“immunoadhesin”), such a fusion could be to the Fc region of an IgGmolecule. The Ig fusions preferably include the substitution of asoluble (transmembrane domain deleted or inactivated) form of a 24P4C12polypeptide in place of at least one variable region within an Igmolecule. In a preferred embodiment, the immunoglobulin fusion includesthe hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of anIgGI molecule. For the production of immunoglobulin fusions see, e.g.,U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

III.D.) Uses of 24P4C12-Related Proteins

The proteins of the invention have a number of different specific uses.As 24P4C12 is highly expressed in prostate and other cancers,24P4C12-related proteins are used in methods that assess the status of24P4C12 gene products in normal versus cancerous tissues, therebyelucidating the malignant phenotype. Typically, polypeptides fromspecific regions of a 24P4C12 protein are used to assess the presence ofperturbations (such as deletions, insertions, point mutations etc.) inthose regions (such as regions containing one or more motifs). Exemplaryassays utilize antibodies or T cells targeting 24P4C12-related proteinscomprising the amino acid residues of one or more of the biologicalmotifs contained within a 24P4C12 polypeptide sequence in order toevaluate the characteristics of this region in normal versus canceroustissues or to elicit an immune response to the epitope. Alternatively,24P4C12-related proteins that contain the amino acid residues of one ormore of the biological motifs in a 24P4C12 protein are used to screenfor factors that interact with that region of 24P4C12.

24P4C12 protein fragments/subsequences are particularly useful ingenerating and characterizing domain-specific antibodies (e.g.,antibodies recognizing an extracellular or intracellular epitope of a24P4C12 protein), for identifying agents or cellular factors that bindto 24P4C12 or a particular structural domain thereof, and in varioustherapeutic and diagnostic contexts, including but not limited todiagnostic assays, cancer vaccines and methods of preparing suchvaccines.

Proteins encoded by the 24P4C12 genes, or by analogs, homologs orfragments thereof, have a variety of uses, including but not limited togenerating antibodies and in methods for identifying ligands and otheragents and cellular constituents that bind to a 24P4C12 gene product.Antibodies raised against a 24P4C12 protein or fragment thereof areuseful in diagnostic and prognostic assays, and imaging methodologies inthe management of human cancers characterized by expression of 24P4C12protein, such as those listed in Table I. Such antibodies can beexpressed intracellularly and used in methods of treating patients withsuch cancers. 24P4C12-related nucleic acids or proteins are also used ingenerating HTL or CTL responses.

Various immunological assays useful for the detection of 24P4C12proteins are used, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Antibodies can be labeled and used asimmunological imaging reagents capable of detecting 24P4C12-expressingcells (e.g., in radioscintigraphic imaging methods). 24P4C12 proteinsare also particularly useful in generating cancer vaccines, as furtherdescribed herein.

IV.) 24P4C12 ANTIBODIES

Another aspect of the invention provides antibodies that bind to24P4C12-related proteins. Preferred antibodies specifically bind to a24P4C12-related protein and do not bind (or bind weakly) to peptides orproteins that are not 24P4C12-related proteins. For example, antibodiesthat bind 24P4C12 can bind 24P4C12-related proteins such as the homologsor analogs thereof.

24P4C12 antibodies of the invention are particularly useful in cancer(see, e.g., Table I) diagnostic and prognostic assays, and imagingmethodologies. Similarly, such antibodies are useful in the treatment,diagnosis, and/or prognosis of other cancers, to the extent 24P4C12 isalso expressed or overexpressed in these other cancers. Moreover,intracellularly expressed antibodies (e.g., single chain antibodies) aretherapeutically useful in treating cancers in which the expression of24P4C12 is involved, such as advanced or metastatic prostate cancers.

The invention also provides various immunological assays useful for thedetection and quantification of 24P4C12 and mutant 24P4C12-relatedproteins. Such assays can comprise one or more 24P4C12 antibodiescapable of recognizing and binding a 24P4C12-related protein, asappropriate. These assays are performed within various immunologicalassay formats well known in the art, including but not limited tovarious types of radioimmunoassays, enzyme-linked immunosorbent assays(ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.

Immunological non-antibody assays of the invention also comprise T cellimmunogenicity assays (inhibitory or stimulatory) as well as majorhistocompatibility complex (MHC) binding assays.

In addition, immunological imaging methods capable of detecting prostatecancer and other cancers expressing 24P4C12 are also provided by theinvention, including but not limited to radioscintigraphic imagingmethods using labeled 24P4C12 antibodies. Such assays are clinicallyuseful in the detection, monitoring, and prognosis of 24P4C12 expressingcancers such as prostate cancer.

24P4C12 antibodies are also used in methods for purifying a24P4C12-related protein and for isolating 24P4C12 homologues and relatedmolecules. For example, a method of purifying a 24P4C12-related proteincomprises incubating a 24P4C12 antibody, which has been coupled to asolid matrix, with a lysate or other solution containing a24P4C12-related protein under conditions that permit the 24P4C12antibody to bind to the 24P4C12-related protein; washing the solidmatrix to eliminate impurities; and eluting the 24P4C12-related proteinfrom the coupled antibody. Other uses of 24P4C12 antibodies inaccordance with the invention include generating anti-idiotypicantibodies that mimic a 24P4C12 protein.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies can be prepared by immunizing a suitablemammalian host using a 24P4C12-related protein, peptide, or fragment, inisolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSHPress, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold SpringHarbor Press, NY (1989)). In addition, fusion proteins of 24P4C12 canalso be used, such as a 24P4C12 GST-fusion protein. In a particularembodiment, a GST fusion protein comprising all or most of the aminoacid sequence of FIG. 2 or FIG. 3 is produced, then used as an immunogento generate appropriate antibodies. In another embodiment, a24P4C12-related protein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used(with or without purified 24P4C12-related protein or 24P4C12 expressingcells) to generate an immune response to the encoded immunogen (forreview, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

The amino acid sequence of a 24P4C12 protein as shown in FIG. 2 or FIG.3 can be analyzed to select specific regions of the 24P4C12 protein forgenerating antibodies. For example, hydrophobicity and hydrophilicityanalyses of a 24P4C12 amino acid sequence are used to identifyhydrophilic regions in the 24P4C12 structure. Regions of a 24P4C12protein that show immunogenic structure, as well as other regions anddomains, can readily be identified using various other methods known inthe art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg,Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can begenerated using the method of Hopp, T. P. and Woods, K. R., 1981, Proc.Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can begenerated using the method of Kyte, J. and Doolittle, R.F., 1982, J.Mol. Biol. 157:105-132. Percent (%)Accessible Residues profiles can begenerated using the method of Janin J., 1979, Nature 277:491-492.Average Flexibility profiles can be generated using the method ofBhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res.32:242-255. Beta-turn profiles can be generated using the method ofDeleage, G., Roux B., 1987, Protein Engineering 1:289-294. Thus, eachregion identified by any of these programs or methods is within thescope of the present invention. Methods for the generation of 24P4C12antibodies are further illustrated by way of the examples providedherein. Methods for preparing a protein or polypeptide for use as animmunogen are well known in the art. Also well known in the art aremethods for preparing immunogenic conjugates of a protein with acarrier, such as BSA, KLH or other carrier protein. In somecircumstances, direct conjugation using, for example, carbodiimidereagents are used; in other instances linking reagents such as thosesupplied by Pierce Chemical Co., Rockford, Ill., are effective.Administration of a 24P4C12 immunogen is often conducted by injectionover a suitable time period and with use of a suitable adjuvant, as isunderstood in the art. During the immunization schedule, titers ofantibodies can be taken to determine adequacy of antibody formation.

24P4C12 monoclonal antibodies can be produced by various means wellknown in the art. For example, immortalized cell lines that secrete adesired monoclonal antibody are prepared using the standard hybridomatechnology of Kohler and Milstein or modifications that immortalizeantibody-producing B cells, as is generally known. Immortalized celllines that secrete the desired antibodies are screened by immunoassay inwhich the antigen is a 24P4C12-related protein. When the appropriateimmortalized cell culture is identified, the cells can be expanded andantibodies produced either from in vitro cultures or from ascites fluid.

The antibodies or fragments of the invention can also be produced, byrecombinant means. Regions that bind specifically to the desired regionsof a 24P4C12 protein can also be produced in the context of chimeric orcomplementarity-determining region (CDR) grafted antibodies of multiplespecies origin. Humanized or human 24P4C12 antibodies can also beproduced, and are preferred for use in therapeutic contexts. Methods forhumanizing murine and other non-human antibodies, by substituting one ormore of the non-human antibody CDRs for corresponding human antibodysequences, are well known (see for example, Jones et al., 1986, Nature321: 522-525; Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen etal., 1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc.Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151:2296.

Methods for producing fully human monoclonal antibodies include phagedisplay and transgenic methods (for review, see Vaughan et al., 1998,Nature Biotechnology 16: 535-539). Fully human 24P4C12 monoclonalantibodies can be generated using cloning technologies employing largehuman Ig gene combinatorial libraries (i.e., phage display) (Griffithsand Hoogenboom, Building an in vitro immune system: human antibodiesfrom phage display libraries. In: Protein Engineering of AntibodyMolecules for Prophylactic and Therapeutic Applications in Man, Clark,M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, HumanAntibodies from combinatorial libraries. Id., pp 65-82). Fully human24P4C12 monoclonal antibodies can also be produced using transgenic miceengineered to contain human immunoglobulin gene loci as described in PCTPatent Application WO98/24893, Kucherlapati and Jakobovits et al.,published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest.Drugs 7(4): 607-614; U.S. Pat. Nos. 6,162,963 issued 19 Dec. 2000;6,150,584 issued 12 Nov. 2000; and, 6,114598 issued 5 Sep. 2000). Thismethod avoids the in vitro manipulation required with phage displaytechnology and efficiently produces high affinity authentic humanantibodies.

Reactivity of 24P4C12 antibodies with a 24P4C12-related protein can beestablished by a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,24P4C12-related proteins, 24P4C12-expressing cells or extracts thereof.A 24P4C12 antibody or fragment thereof can be labeled with a detectablemarker or conjugated to a second molecule. Suitable detectable markersinclude, but are not limited to, a radioisotope, a fluorescent compound,a bioluminescent compound, chemiluminescent compound, a metal chelatoror an enzyme. Further, bi-specific antibodies specific for two or more24P4C12 epitopes are generated using methods generally known in the art.Homodimeric antibodies can also be generated by cross-linking techniquesknown in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).

V.) 24P4C12 CELLULAR IMMUNE RESPONSES

The mechanism by which T cells recognize antigens has been delineated.Efficacious peptide epitope vaccine compositions of the invention inducea therapeutic or prophylactic immune responses in very broad segments ofthe world-wide population. For an understanding of the value andefficacy of compositions of the invention that induce cellular immuneresponses, a brief review of immunology-related technology is provided.

A complex of an HLA molecule and a peptidic antigen acts as the ligandrecognized by HLA-restricted T cells (Buus, S. et al., Cell 147:1071,1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. andBodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev.Immunol. 11:403, 1993). Through the study of single amino acidsubstituted antigen analogs and the sequencing of endogenously bound,naturally processed peptides, critical residues that correspond tomotifs required for specific binding to HLA antigen molecules have beenidentified and are set forth in Table IV (see also, e.g., Southwood, etal., J. Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics41:178, 1995; Rammensee et al., SYFPEITHI, access via World Wide Web atURL (134.2.96.221/scripts.hlaserver.dll/home.htm); Sette, A. and Sidney,J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin.Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol.4:79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994;Ruppert et al., Cell 74:929-937, 1993; Kondo et al., J. Immunol.155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490, 1996;Sidney et al., Human Immunol. 45:79-93, 1996; Sette, A. and Sidney, J.Immunogenetics 1999 November; 50(3-4):201-12, Review).

Furthermore, x-ray crystallographic analyses of HLA-peptide complexeshave revealed pockets within the peptide binding cleft/groove of HLAmolecules which accommodate, in an allele-specific mode, residues borneby peptide ligands; these residues in turn determine the HLA bindingcapacity of the peptides in which they are present. (See, e.g., Madden,D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203,1996; Fremont et al., Immunity 8:305, 1998; Stern et al., Structure2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H.et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci.USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M.L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927,1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D.C., J. Mol.Biol. 219:277, 1991.)

Accordingly, the definition of class I and class II allele-specific HLAbinding motifs, or class I or class II supermotifs allows identificationof regions within a protein that are correlated with binding toparticular HLA antigen(s).

Thus, by a process of HLA motif identification, candidates forepitope-based vaccines have been identified; such candidates can befurther evaluated by HLA-peptide binding assays to determine bindingaffinity and/or the time period of association of the epitope and itscorresponding HLA molecule. Additional confirmatory work can beperformed to select, amongst these vaccine candidates, epitopes withpreferred characteristics in terms of population coverage, and/orimmunogenicity.

Various strategies can be utilized to evaluate cellular immunogenicity,including:

1) Evaluation of primary T cell cultures from normal individuals (see,e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. etal., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J.Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1,1998). This procedure involves the stimulation of peripheral bloodlymphocytes (PBL) from normal subjects with a test peptide in thepresence of antigen presenting cells in vitro over a period of severalweeks. T cells specific for the peptide become activated during thistime and are detected using, e.g., a lymphokine- or ⁵¹Cr-release assayinvolving peptide sensitized target cells.

2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. etal., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol.8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997). Forexample, in such methods peptides in incomplete Freund's adjuvant areadministered subcutaneously to HLA transgenic mice, Several weeksfollowing immunization, splenocytes are removed and cultured in vitro inthe presence of test peptide for approximately one week.Peptide-specific T cells are detected using, e.g., a ⁵¹Cr-release assayinvolving peptide sensitized target cells and target cells expressingendogenously generated antigen.

3) Demonstration of recall T cell responses from immune individuals whohave been either effectively vaccinated and/or from chronically illpatients (see, e.g., Rehermann, B. et al., J. Exp. Med. 181:1047, 1995;Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin.Invest 100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 159:1648,1997; Diepolder, H. M. et al., J. Virol. 71:6011, 1997). Accordingly,recall responses are detected by culturing PBL from subjects that havebeen exposed to the antigen due to disease and thus have generated animmune response “naturally”, or from patients who were vaccinatedagainst the antigen. PBL from subjects are cultured in vitro for 1-2weeks in the presence of test peptide plus antigen presenting cells(APC) to allow activation of “memory” T cells, as compared to “naive” Tcells. At the end of the culture period, T cell activity is detectedusing assays including ⁵¹Cr release involving peptide-sensitizedtargets, T cell proliferation, or lymphokine release.

VI.) 24P4C12 TRANSGENIC ANIMALS

Nucleic acids that encode a 24P4C12-related protein can also be used togenerate either transgenic animals or “knock out” animals that, in turn,are useful in the development and screening of therapeutically usefulreagents. In accordance with established techniques, cDNA encoding24P4C12 can be used to clone genomic DNA that encodes 24P4C12. Thecloned genomic sequences can then be used to generate transgenic animalscontaining cells that express DNA that encode 24P4C12. Methods forgenerating transgenic animals, particularly animals such as mice orrats, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 issued 12 Apr. 1988, and 4,870,009issued 26 Sep. 1989. Typically, particular cells would be targeted for24P4C12 transgene incorporation with tissue-specific enhancers.

Transgenic animals that include a copy of a transgene encoding 24P4C12can be used to examine the effect of increased expression of DNA thatencodes 24P4C12. Such animals can be used as tester animals for reagentsthought to confer protection from, for example, pathological conditionsassociated with its overexpression. In accordance with this aspect ofthe invention, an animal is treated with a reagent and a reducedincidence of a pathological condition, compared to untreated animalsthat bear the transgene, would indicate a potential therapeuticintervention for the pathological condition.

Alternatively, non-human homologues of 24P4C12 can be used to constructa 24P4C12 “knock out” animal that has a defective or altered geneencoding 24P4C12 as a result of homologous recombination between theendogenous gene encoding 24P4C12 and altered genomic DNA encoding24P4C12 introduced into an embryonic cell of the animal. For example,cDNA that encodes 24P4C12 can be used to clone genomic DNA encoding24P4C12 in accordance with established techniques. A portion of thegenomic DNA encoding 24P4C12 can be deleted or replaced with anothergene, such as a gene encoding a selectable marker that can be used tomonitor integration. Typically, several kilobases of unaltered flankingDNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g.,Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected(see, e.g., Li et al., Cell, 69:915 (1992)). The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras (see, e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152). A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal, and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knock out animals can becharacterized, for example, for their ability to defend against certainpathological conditions or for their development of pathologicalconditions due to absence of a 24P4C12 polypeptide.

VII.) METHODS FOR THE DETECTION OF 24P4C12

Another aspect of the present invention relates to methods for detecting24P4C12 polynucleotides and 24P4C12-related proteins, as well as methodsfor identifying a cell that expresses 24P4C12. The expression profile of24P4C12 makes it a diagnostic marker for metastasized disease.Accordingly, the status of 24P4C12 gene products provides informationuseful for predicting a variety of factors including susceptibility toadvanced stage disease, rate of progression, and/or tumoraggressiveness. As discussed in detail herein, the status of 24P4C12gene products in patient samples can be analyzed by a variety protocolsthat are well known in the art including immunohistochemical analysis,the variety of Northern blotting techniques including in situhybridization, RT-PCR analysis (for example on laser capturemicro-dissected samples), Western blot analysis and tissue arrayanalysis.

More particularly, the invention provides assays for the detection of24P4C12 polynucleotides in a biological sample, such as serum, bone,prostate, and other tissues, urine, semen, cell preparations, and thelike. Detectable 24P4C12 polynucleotides include, for example, a 24P4C12gene or fragment thereof, 24P4C12 mRNA, alternative splice variant24P4C12 mRNAs, and recombinant DNA or RNA molecules that contain a24P4C12 polynucleotide. A number of methods for amplifying and/ordetecting the presence of 24P4C12 polynucleotides are well known in theart and can be employed in the practice of this aspect of the invention.

In one embodiment, a method for detecting a 24P4C12 mRNA in a biologicalsample comprises producing cDNA from the sample by reverse transcriptionusing at least one primer; amplifying the cDNA so produced using a24P4C12 polynucleotides as sense and antisense primers to amplify24P4C12 cDNAs therein; and detecting the presence of the amplified24P4C12 cDNA. Optionally, the sequence of the amplified 24P4C12 cDNA canbe determined.

In another embodiment, a method of detecting a 24P4C12 gene in abiological sample comprises first isolating genomic DNA from the sample;amplifying the isolated genomic DNA using 24P4C12 polynucleotides assense and antisense primers; and detecting the presence of the amplified24P4C12 gene. Any number of appropriate sense and antisense probecombinations can be designed from a 24P4C12 nucleotide sequence (see,e.g., FIG. 2) and used for this purpose.

The invention also provides assays for detecting the presence of a24P4C12 protein in a tissue or other biological sample such as serum,semen, bone, prostate, urine, cell preparations, and the like. Methodsfor detecting a 24P4C12-related protein are also well known and include,for example, immunoprecipitation, immunohistochemical analysis, Westernblot analysis, molecular binding assays, ELISA, ELIFA and the like. Forexample, a method of detecting the presence of a 24P4C12-related proteinin a biological sample comprises first contacting the sample with a24P4C12 antibody, a 24P4C12-reactive fragment thereof, or a recombinantprotein containing an antigen-binding region of a 24P4C12 antibody; andthen detecting the binding of 24P4C12-related protein in the sample.

Methods for identifying a cell that expresses 24P4C12 are also withinthe scope of the invention. In one embodiment, an assay for identifyinga cell that expresses a 24P4C12 gene comprises detecting the presence of24P4C12 mRNA in the cell. Methods for the detection of particular mRNAsin cells are well known and include, for example, hybridization assaysusing complementary DNA probes (such as in situ hybridization usinglabeled 24P4C12 riboprobes, Northern blot and related techniques) andvarious nucleic acid amplification assays (such as RT-PCR usingcomplementary primers specific for 24P4C12, and other amplification typedetection methods, such as, for example, branched DNA, SISBA, TMA andthe like). Alternatively, an assay for identifying a cell that expressesa 24P4C12 gene comprises detecting the presence of 24P4C12-relatedprotein in the cell or secreted by the cell. Various methods for thedetection of proteins are well known in the art and are employed for thedetection of 24P4C12-related proteins and cells that express24P4C12-related proteins.

24P4C12 expression analysis is also useful as a tool for identifying andevaluating agents that modulate 24P4C12 gene expression. For example,24P4C12 expression is significantly upregulated in prostate cancer, andis expressed in cancers of the tissues listed in Table I. Identificationof a molecule or biological agent that inhibits 24P4C12 expression orover-expression in cancer cells is of therapeutic value. For example,such an agent can be identified by using a screen that quantifies24P4C12 expression by RT-PCR, nucleic acid hybridization or antibodybinding.

VIII.) METHODS FOR MONITORING THE STATUS OF 24P4C12-RELATED GENES ANDTHEIR PRODUCTS

Oncogenesis is known to be a multistep process where cellular growthbecomes progressively dysregulated and cells progress from a normalphysiological state to precancerous and then cancerous states (see,e.g., Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs et al.,Cancer Surv. 23: 19-32 (1995)). In this context, examining a biologicalsample for evidence of dysregulated cell growth (such as aberrant24P4C12 expression in cancers) allows for early detection of suchaberrant physiology, before a pathologic state such as cancer hasprogressed to a stage that therapeutic options are more limited and orthe prognosis is worse. In such examinations, the status of 24P4C12 in abiological sample of interest can be compared, for example, to thestatus of 24P4C12 in a corresponding normal sample (e.g. a sample fromthat individual or alternatively another individual that is not affectedby a pathology). An alteration in the status of 24P4C12 in thebiological sample (as compared to the normal sample) provides evidenceof dysregulated cellular growth. In addition to using a biologicalsample that is not affected by a pathology as a normal sample, one canalso use a predetermined normative value such as a predetermined normallevel of mRNA expression (see, e.g., Grever et al., J. Comp. Neurol.1996 Dec. 9; 376(2): 306-14 and U.S. Pat. No. 5,837,501) to compare24P4C12 status in a sample.

The term “status” in this context is used according to its art acceptedmeaning and refers to the condition or state of a gene and its products.Typically, skilled artisans use a number of parameters to evaluate thecondition or state of a gene and its products. These include, but arenot limited to the location of expressed gene products (including thelocation of 24P4C12 expressing cells) as well as the level, andbiological activity of expressed gene products (such as 24P4C12 mRNA,polynucleotides and polypeptides). Typically, an alteration in thestatus of 24P4C12 comprises a change in the location of 24P4C12 and/or24P4C12 expressing cells and/or an increase in 24P4C12 mRNA and/orprotein expression.

24P4C12 status in a sample can be analyzed by a number of means wellknown in the art, including without limitation, immunohistochemicalanalysis, in situ hybridization, RT-PCR analysis on laser capturemicro-dissected samples, Western blot analysis, and tissue arrayanalysis. Typical protocols for evaluating the status of a 24P4C12 geneand gene products are found, for example in Ausubel et al. eds., 1995,Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4(Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus,the status of 24P4C12 in a biological sample is evaluated by variousmethods utilized by skilled artisans including, but not limited togenomic Southern analysis (to examine, for example perturbations in a24P4C12 gene), Northern analysis and/or PCR analysis of 24P4C12 mRNA (toexamine, for example alterations in the polynucleotide sequences orexpression levels of 24P4C12 mRNAs), and, Western and/orimmunohistochemical analysis (to examine, for example alterations inpolypeptide sequences, alterations in polypeptide localization within asample, alterations in expression levels of 24P4012 proteins and/orassociations of 24P4C12 proteins with polypeptide binding partners).Detectable 24P4C12 polynucleotides include, for example, a 24P4C12 geneor fragment thereof, 24P4C12 mRNA, alternative splice variants, 24P4C12mRNAs, and recombinant DNA or RNA molecules containing a 24P4C12polynucleotide.

The expression profile of 24P4C12 makes it a diagnostic marker for localand/or metastasized disease, and provides information on the growth oroncogenic potential of a biological sample. In particular, the status of24P4C12 provides information useful for predicting susceptibility toparticular disease stages, progression, and/or tumor aggressiveness. Theinvention provides methods and assays for determining 24P4C12 status anddiagnosing cancers that express 24P4C12, such as cancers of the tissueslisted in Table I. For example, because 24P4C12 mRNA is so highlyexpressed in prostate and other cancers relative to normal prostatetissue, assays that evaluate the levels of 24P4C12 mRNA transcripts orproteins in a biological sample can be used to diagnose a diseaseassociated with 24P4C12 dysregulation, and can provide prognosticinformation useful in defining appropriate therapeutic options.

The expression status of 24P4C12 provides information including thepresence, stage and location of dysplastic, precancerous and cancerouscells, predicting susceptibility to various stages of disease, and/orfor gauging tumor aggressiveness. Moreover, the expression profile makesit useful as an imaging reagent for metastasized disease. Consequently,an aspect of the invention is directed to the various molecularprognostic and diagnostic methods for examining the status of 24P4C12 inbiological samples such as those from individuals suffering from, orsuspected of suffering from a pathology characterized by dysregulatedcellular growth, such as cancer.

As described above, the status of 24P4C12 in a biological sample can beexamined by a number of well-known procedures in the art. For example,the status of 24P4C12 in a biological sample taken from a specificlocation in the body can be examined by evaluating the sample for thepresence or absence of 24P4C12 expressing cells (e.g. those that express24P4C12 mRNAs or proteins). This examination can provide evidence ofdysregulated cellular growth, for example, when 24P4C12-expressing cellsare found in a biological sample that does not normally contain suchcells (such as a lymph node), because such alterations in the status of24P4C12 in a biological sample are often associated with dysregulatedcellular growth. Specifically, one indicator of dysregulated cellulargrowth is the metastases of cancer cells from an organ of origin (suchas the prostate) to a different area of the body (such as a lymph node).In this context, evidence of dysregulated cellular growth is importantfor example because occult lymph node metastases can be detected in asubstantial proportion of patients with prostate cancer, and suchmetastases are associated with known predictors of disease progression(see, e.g., Murphy et al., Prostate 42(4): 315-317 (2000);Su et al.,Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., J Urol 1995August 154 (2 Pt 1):474-8).

In one aspect, the invention provides methods for monitoring 24P4C12gene products by determining the status of 24P4C12 gene productsexpressed by cells from an individual suspected of having a diseaseassociated with dysregulated cell growth (such as hyperplasia or cancer)and then comparing the status so determined to the status of 24P4C12gene products in a corresponding normal sample. The presence of aberrant24P4C12 gene products in the test sample relative to the normal sampleprovides an indication of the presence of dysregulated cell growthwithin the cells of the individual.

In another aspect, the invention provides assays useful in determiningthe presence of cancer in an individual, comprising detecting asignificant increase in 24P4C12 mRNA or protein expression in a testcell or tissue sample relative to expression levels in the correspondingnormal cell or tissue. The presence of 24P4C12 mRNA can, for example, beevaluated in tissues including but not limited to those listed in TableI. The presence of significant 24P4C12 expression in any of thesetissues is useful to indicate the emergence, presence and/or severity ofa cancer, since the corresponding normal tissues do not express 24P4C12mRNA or express it at lower levels.

In a related embodiment, 24P4C12 status is determined at the proteinlevel rather than at the nucleic acid level. For example, such a methodcomprises determining the level of 24P4C12 protein expressed by cells ina test tissue sample and comparing the level so determined to the levelof 24P4C12 expressed in a corresponding normal sample. In oneembodiment, the presence of 24P4C12 protein is evaluated, for example,using immunohistochemical methods. 24P4C12 antibodies or bindingpartners capable of detecting 24P4C12 protein expression are used in avariety of assay formats well known in the art for this purpose.

In a further embodiment, one can evaluate the status of 24P4C12nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules. Theseperturbations can include insertions, deletions, substitutions and thelike. Such evaluations are useful because perturbations in thenucleotide and amino acid sequences are observed in a large number ofproteins associated with a growth dysregulated phenotype (see, e.g.,Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, amutation in the sequence of 24P4C12 may be indicative of the presence orpromotion of a tumor. Such assays therefore have diagnostic andpredictive value where a mutation in 24P4C12 indicates a potential lossof function or increase in tumor growth.

A wide variety of assays for observing perturbations in nucleotide andamino acid sequences are well known in the art. For example, the sizeand structure of nucleic acid or amino acid sequences of 24P4C12 geneproducts are observed by the Northern, Southern, Western, PCR and DNAsequencing protocols discussed herein. In addition, other methods forobserving perturbations in nucleotide and amino acid sequences such assingle strand conformation polymorphism analysis are well known in theart (see, e.g., U.S. Pat. Nos. 5,382,510 issued 7 Sep. 1999, and5,952,170 issued 17 Jan. 1995).

Additionally, one can examine the methylation status of a 24P4C12 genein a biological sample. Aberrant demethylation and/or hypermethylationof CpG islands in gene 5′ regulatory regions frequently occurs inimmortalized and transformed cells, and can result in altered expressionof various genes. For example, promoter hypermethylation of the pi-classglutathione S-transferase (a protein expressed in normal prostate butnot expressed in >90% of prostate carcinomas) appears to permanentlysilence transcription of this gene and is the most frequently detectedgenomic alteration in prostate carcinomas (De Marzo et al., Am. J.Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration ispresent in at least 70% of cases of high-grade prostatic intraepithelialneoplasia (PIN) (Brooks et al., Cancer Epidemiol. Biomarkers Prev.,1998, 7:531-536). In another example, expression of the LAGE-I tumorspecific gene (which is not expressed in normal prostate but isexpressed in 25-50% of prostate cancers) is induced by deoxy-azacytidinein lymphoblastoid cells, suggesting that tumoral expression is due todemethylation (Lethe et al., Int. J. Cancer 76(6): 903-908 (1998)). Avariety of assays for examining methylation status of a gene are wellknown in the art. For example, one can utilize, in Southernhybridization approaches, methylation-sensitive restriction enzymes thatcannot cleave sequences that contain methylated CpG sites to assess themethylation status of CpG islands. In addition, MSP (methylationspecific PCR) can rapidly profile the methylation status of all the CpGsites present in a CpG island of a given gene. This procedure involvesinitial modification of DNA by sodium bisulfite (which will convert allunmethylated cytosines to uracil) followed by amplification usingprimers specific for methylated versus unmethylated DNA. Protocolsinvolving methylation interference can also be found for example inCurrent Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel etal. eds., 1995.

Gene amplification is an additional method for assessing the status of24P4C12. Gene amplification is measured in a sample directly, forexample, by conventional Southern blotting or Northern blotting toquantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad.Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies are employed thatrecognize specific duplexes, including DNA duplexes, RNA duplexes, andDNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turnare labeled and the assay carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Biopsied tissue or peripheral blood can be conveniently assayed for thepresence of cancer cells using for example, Northern, dot blot or RT-PCRanalysis to detect 24P4C12 expression. The presence of RT-PCRamplifiable 24P4C12 mRNA provides an indication of the presence ofcancer. RT-PCR assays are well known in the art. RT-PCR detection assaysfor tumor cells in peripheral blood are currently being evaluated foruse in the diagnosis and management of a number of human solid tumors.In the prostate cancer field, these include RT-PCR assays for thedetection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol.Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000;Heston et al., 1995, Clin. Chem. 41:1687-1688).

A further aspect of the invention is an assessment of the susceptibilitythat an individual has for developing cancer. In one embodiment, amethod for predicting susceptibility to cancer comprises detecting24P4C12 mRNA or 24P4C12 protein in a tissue sample, its presenceindicating susceptibility to cancer, wherein the degree of 24P4C12 mRNAexpression correlates to the degree of susceptibility. In a specificembodiment, the presence of 24P4C12 in prostate or other tissue isexamined, with the presence of 24P4C12 in the sample providing anindication of prostate cancer susceptibility (or the emergence orexistence of a prostate tumor). Similarly, one can evaluate theintegrity 24P4C12 nucleotide and amino acid sequences in a biologicalsample, in order to identify perturbations in the structure of thesemolecules such as insertions, deletions, substitutions and the like. Thepresence of one or more perturbations in 24P4C12 gene products in thesample is an indication of cancer susceptibility (or the emergence orexistence of a tumor).

The invention also comprises methods for gauging tumor aggressiveness.In one embodiment, a method for gauging aggressiveness of a tumorcomprises determining the level of 24P4C12 mRNA or 24P4C12 proteinexpressed by tumor cells, comparing the level so determined to the levelof 24P4C12 mRNA or 24P4C12 protein expressed in a corresponding normaltissue taken from the same individual or a normal tissue referencesample, wherein the degree of 24P4C12 mRNA or 24P4C12 protein expressionin the tumor sample relative to the normal sample indicates the degreeof aggressiveness. In a specific embodiment, aggressiveness of a tumoris evaluated by determining the extent to which 24P4C12 is expressed inthe tumor cells, with higher expression levels indicating moreaggressive tumors. Another embodiment is the evaluation of the integrityof 24P4C12 nucleotide and amino acid sequences in a biological sample,in order to identify perturbations in the structure of these moleculessuch as insertions, deletions, substitutions and the like. The presenceof one or more perturbations indicates more aggressive tumors.

Another embodiment of the invention is directed to methods for observingthe progression of a malignancy in an individual over time. In oneembodiment, methods for observing the progression of a malignancy in anindividual over time comprise determining the level of 24P4C12 mRNA or24P4C12 protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of 24P4C12 mRNA or 24P4C12 proteinexpressed in an equivalent tissue sample taken from the same individualat a different time, wherein the degree of 24P4C12 mRNA or 24P4C12protein expression in the tumor sample over time provides information onthe progression of the cancer. In a specific embodiment, the progressionof a cancer is evaluated by determining 24P4C12 expression in the tumorcells over time, where increased expression over time indicates aprogression of the cancer. Also, one can evaluate the integrity 24P4C12nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules such asinsertions, deletions, substitutions and the like, where the presence ofone or more perturbations indicates a progression of the cancer.

The above diagnostic approaches can be combined with any one of a widevariety of prognostic and diagnostic protocols known in the art. Forexample, another embodiment of the invention is directed to methods forobserving a coincidence between the expression of 24P4C12 gene and24P4C12 gene products (or perturbations in 24P4C12 gene and 24P4C12 geneproducts) and a factor that is associated with malignancy, as a meansfor diagnosing and prognosticating the status of a tissue sample. A widevariety of factors associated with malignancy can be utilized, such asthe expression of genes associated with malignancy (e.g. PSA, PSCA andPSM expression for prostate cancer etc.) as well as gross cytologicalobservations (see, e.g., Bocking et al., 1984, Anal. Quant. Cytol.6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al.,1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg.Pathol. 23(8):918-24). Methods for observing a coincidence between theexpression of 24P4C12 gene and 24P4C12 gene products (or perturbationsin 24P4C12 gene and 24P4C12 gene products) and another factor that isassociated with malignancy are useful, for example, because the presenceof a set of specific factors that coincide with disease providesinformation crucial for diagnosing and prognosticating the status of atissue sample.

In one embodiment, methods for observing a coincidence between theexpression of 24P4C12 gene and 24P4C12 gene products (or perturbationsin 24P4C12 gene and 24P4C12 gene products) and another factor associatedwith malignancy entails detecting the overexpression of 24P4C12 mRNA orprotein in a tissue sample, detecting the overexpression of PSA mRNA orprotein in a tissue sample (or PSCA or PSM expression), and observing acoincidence of 24P4C12 mRNA or protein and PSA mRNA or proteinoverexpression (or PSCA or PSM expression). In a specific embodiment,the expression of 24P4C12 and PSA mRNA in prostate tissue is examined,where the coincidence of 24P4C12 and PSA mRNA overexpression in thesample indicates the existence of prostate cancer, prostate cancersusceptibility or the emergence or status of a prostate tumor.

Methods for detecting and quantifying the expression of 24P4C12 mRNA orprotein are described herein, and standard nucleic acid and proteindetection and quantification technologies are well known in the art.Standard methods for the detection and quantification of 24P4C12 mRNAinclude in situ hybridization using labeled 24P4C12 riboprobes, Northernblot and related techniques using 24P4C12 polynucleotide probes, RT-PCRanalysis using primers specific for 24P4C12, and other amplificationtype detection methods, such as, for example, branched DNA, SISBA, TMAand the like. In a specific embodiment, semi-quantitative RT-PCR is usedto detect and quantify 24P4C12 mRNA expression. Any number of primerscapable of amplifying 24P4C12 can be used for this purpose, includingbut not limited to the various primer sets specifically describedherein. In a specific embodiment, polyclonal or monoclonal antibodiesspecifically reactive with the wild-type 24P4C12 protein can be used inan immunohistochemical assay of biopsied tissue.

IX.) IDENTIFICATION OF MOLECULES THAT INTERACT WITH 24P4C12

The 24P4C12 protein and nucleic acid sequences disclosed herein allow askilled artisan to identify proteins, small molecules and other agentsthat interact with 24P4C12, as well as pathways activated by 24P4C12 viaany one of a variety of art accepted protocols. For example, one canutilize one of the so-called interaction trap systems (also referred toas the “two-hybrid assay”). In such systems, molecules interact andreconstitute a transcription factor which directs expression of areporter gene, whereupon the expression of the reporter gene is assayed.Other systems identify protein-protein interactions in vivo throughreconstitution of a eukaryotic transcriptional activator, see, e.g.,U.S. Pat. Nos. 5,955,280 issued 21 Sep. 1999, 5,925,523 issued 20 July1999, 5,846,722 issued 8 Dec. 1998 and 6,004,746 issued 21 Dec. 1999.Algorithms are also available in the art for genome-based predictions ofprotein function (see, e.g., Marcotte, et al., Nature 402: 4 Nov. 1999,83-86).

Alternatively one can screen peptide libraries to identify moleculesthat interact with 24P4C12 protein sequences. In such methods, peptidesthat bind to 24P4C12 are identified by screening libraries that encode arandom or controlled collection of amino acids. Peptides encoded by thelibraries are expressed as fusion proteins of bacteriophage coatproteins, the bacteriophage particles are then screened against the24P4C12 protein(s).

Accordingly, peptides having a wide variety of uses, such astherapeutic, prognostic or diagnostic reagents, are thus identifiedwithout any prior information on the structure of the expected ligand orreceptor molecule. Typical peptide libraries and screening methods thatcan be used to identify molecules that interact with 24P4C12 proteinsequences are disclosed for example in U.S. Pat. Nos. 5,723,286 issued 3Mar. 1998 and 5,733,731 issued 31 Mar. 1998.

Alternatively, cell lines that express 24P4C12 are used to identifyprotein-protein interactions mediated by 24P4C12. Such interactions canbe examined using immunoprecipitation techniques (see, e.g., Hamilton B.J., et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). 24P4C12protein can be immunoprecipitated from 24P4C12-expressing cell linesusing anti-24P4C12 antibodies. Alternatively, antibodies against His-tagcan be used in a cell line engineered to express fusions of 24P4C12 anda His-tag (vectors mentioned above). The immunoprecipitated complex canbe examined for protein association by procedures such as Westernblotting, ³⁵S-methionine labeling of proteins, protein microsequencing,silver staining and two-dimensional gel electrophoresis.

Small molecules and ligands that interact with 24P4C12 can be identifiedthrough related embodiments of such screening assays. For example, smallmolecules can be identified that interfere with protein function,including molecules that interfere with 24P4C12's ability to mediatephosphorylation and de-phosphorylation, interaction with DNA or RNAmolecules as an indication of regulation of cell cycles, secondmessenger signaling or tumorigenesis. Similarly, small molecules thatmodulate 24P4C12-related ion channel, protein pump, or cellcommunication functions are identified and used to treat patients thathave a cancer that expresses 24P4C12 (see, e.g., Hille, B., IonicChannels of Excitable Membranes 2^(nd) Ed., Sinauer Assoc., Sunderland,Mass., 1992). Moreover, ligands that regulate 24P4C12 function can beidentified based on their ability to bind 24P4C12 and activate areporter construct. Typical methods are discussed for example in U.S.Pat. No. 5,928,868 issued 27 Jul. 1999, and include methods for forminghybrid ligands in which at least one ligand is a small molecule. In anillustrative embodiment, cells engineered to express a fusion protein of24P4C12 and a DNA-binding protein are used to co-express a fusionprotein of a hybrid ligand/small molecule and a cDNA librarytranscriptional activator protein. The cells further contain a reportergene, the expression of which is conditioned on the proximity of thefirst and second fusion proteins to each other, an event that occursonly if the hybrid ligand binds to target sites on both hybrid proteins.Those cells that express the reporter gene are selected and the unknownsmall molecule or the unknown ligand is identified. This method providesa means of identifying modulators, which activate or inhibit 24P4C12.

An embodiment of this invention comprises a method of screening for amolecule that interacts with a 24P4C12 amino acid sequence shown in FIG.2 or FIG. 3, comprising the steps of contacting a population ofmolecules with a 24P4C12 amino acid sequence, allowing the population ofmolecules and the 24P4C12 amino acid sequence to interact underconditions that facilitate an interaction, determining the presence of amolecule that interacts with the 24P4C12 amino acid sequence, and thenseparating molecules that do not interact with the 24P4C12 amino acidsequence from molecules that do. In a specific embodiment, the methodfurther comprises purifying, characterizing and identifying a moleculethat interacts with the 24P4C12 amino acid sequence. The identifiedmolecule can be used to modulate a function performed by 24P4C12. In apreferred embodiment, the 24P4C12 amino acid sequence is contacted witha library of peptides.

X.) THERAPEUTIC METHODS AND COMPOSITIONS

The identification of 24P4C12 as a protein that is normally expressed ina restricted set of tissues, but which is also expressed in prostate andother cancers, opens a number of therapeutic approaches to the treatmentof such cancers. As contemplated herein, 24P4C12 functions as atranscription factor involved in activating tumor-promoting genes orrepressing genes that block tumorigenesis.

Accordingly, therapeutic approaches that inhibit the activity of a24P4C12 protein are useful for patients suffering from a cancer thatexpresses 24P4C12. These therapeutic approaches generally fall into twoclasses. One class comprises various methods for inhibiting the bindingor association of a 24P4C12 protein with its binding partner or withother proteins. Another class comprises a variety of methods forinhibiting the transcription of a 24P4C12 gene or translation of 24P4C12mRNA.

X.A.) Anti-Cancer Vaccines

The invention provides cancer vaccines comprising a 24P4C12-relatedprotein or 24P4C12-related nucleic acid. In view of the expression of24P4C12, cancer vaccines prevent and/or treat 24P4C12-expressing cancerswith minimal or no effects on non-target tissues. The use of a tumorantigen in a vaccine that generates humoral and/or cell-mediated immuneresponses as anti-cancer therapy is well known in the art and has beenemployed in prostate cancer using human PSMA and rodent PAPimmunogens(Hodge et al., 1995, Int. J. Cancer 63:231-237; Fong et al.,1997, J. Immunol. 159:3113-3117).

Such methods can be readily practiced by employing a 24P4C12-relatedprotein, or a 24P4C12-encoding nucleic acid molecule and recombinantvectors capable of expressing and presenting the 24P4C12 immunogen(which typically comprises a number of antibody or T cell epitopes).Skilled artisans understand that a wide variety of vaccine systems fordelivery of immunoreactive epitopes are known in the art (see, e.g.,Heryln et al., Ann Med 1999 February 31(1):66-78; Maruyama et al.,Cancer Immunol Immunother 2000 June 49(3):123-32) Briefly, such methodsof generating an immune response (e.g. humoral and/or cell-mediated) ina mammal, comprise the steps of: exposing the mammal's immune system toan immunoreactive epitope (e.g. an epitope present in a 24P4C12 proteinshown in FIG. 3 or analog or homolog thereof) so that the mammalgenerates an immune response that is specific for that epitope (e.g.generates antibodies that specifically recognize that epitope). In apreferred method, a 24P4C12 immunogen contains a biological motif, seee.g., Tables VIII-XXI and XXII-XLIX, or a peptide of a size range from24P4C12 indicated in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.

The entire 24P4C12 protein, immunogenic regions or epitopes thereof canbe combined and delivered by various means. Such vaccine compositionscan include, for example, lipopeptides (e.g., Vitiello, A. et al., J.Clin. Invest. 95:341, 1995), peptide compositions encapsulated inpoly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge,et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptidecompositions contained in immune stimulating complexes (ISCOMS) (see,e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin ExpImmunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs)(see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988;Tam, J.P., J. Immunol. Methods 196:17-32, 1996), peptides formulated asmultivalent peptides; peptides for use in ballistic delivery systems,typically crystallized peptides, viral delivery vectors (Perkus, M. E.et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p.379,1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. etal., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda,P. K. et al., Virology 175:535, 1990), particles of viral or syntheticorigin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996;Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. etal., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R.,and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al.,Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol.148:1585, 1992; Rack, K. L., Immunol. Today 17:131, 1996), or, naked orparticle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993;Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993;Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S.H. E., ed., p. 423,1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev.Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16,1993). Toxin-targeted delivery technologies, also known as receptormediated targeting, such as those of Avant Immunotherapeutics, Inc.(Needham, Mass.) may also be used.

In patients with 24P4C12-associated cancer, the vaccine compositions ofthe invention can also be used in conjunction with other treatments usedfor cancer, e.g., surgery, chemotherapy, drug therapies, radiationtherapies, etc. including use in combination with immune adjuvants suchas IL-2, IL-12, GM-CSF, and the like.

Cellular Vaccines:

CTL epitopes can be determined using specific algorithms to identifypeptides within 24P4C12 protein that bind corresponding HLA alleles (seee.g., Table IV; Epimer™ and Epimatrix™, Brown University (URLbrown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and, BIMAS,(URL bimas.dcrt.nih.gov/; SYFPEITHI at URLsyfpeithi.bmi-heidelberg.com/). In a preferred embodiment, a 24P4C12immunogen contains one or more amino acid sequences identified usingtechniques well known in the art, such as the sequences shown in TablesVIII-XXI and XXII-XLIX or a peptide of 8, 9, 10 or 11 amino acidsspecified by an HLA Class I motif/supermotif (e.g., Table IV (A), TableIV (D), or Table IV (E)) and/or a peptide of at least 9 amino acids thatcomprises an HLA Class II motif/supermotif (e.g., Table IV (B) or TableIV (C)). As is appreciated in the art, the HLA Class I binding groove isessentially closed ended so that peptides of only a particular sizerange can fit into the groove and be bound, generally HLA Class Iepitopes are 8, 9, 10, or 11 amino acids long. In contrast, the HLAClass II binding groove is essentially open ended; therefore a peptideof about 9 or more amino acids can be bound by an HLA Class II molecule.Due to the binding groove differences between HLA Class I and II, HLAClass I motifs are length specific, i.e., position two of a Class Imotif is the second amino acid in an amino to carboxyl direction of thepeptide. The amino acid positions in a Class II motif are relative onlyto each other, not the overall peptide, i.e., additional amino acids canbe attached to the amino and/or carboxyl termini of a motif-bearingsequence. HLA Class II epitopes are often 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long, or longer than25 amino acids.

Antibody-Based Vaccines

A wide variety of methods for generating an immune response in a mammalare known in the art (for example as the first step in the generation ofhybridomas). Methods of generating an immune response in a mammalcomprise exposing the mammal's immune system to an immunogenic epitopeon a protein (e.g. a 24P4C12 protein) so that an immune response isgenerated. A typical embodiment consists of a method for generating animmune response to 24P4C12 in a host, by contacting the host with asufficient amount of at least one 24P4C12 B cell or cytotoxic T-cellepitope or analog thereof; and at least one periodic interval thereafterre-contacting the host with the 24P4C12 B cell or cytotoxic T-cellepitope or analog thereof. A specific embodiment consists of a method ofgenerating an immune response against a 24P4C12-related protein or aman-made multiepitopic peptide comprising: administering 24P4C12immunogen (e.g. a 24P4C12 protein or a peptide fragment thereof, a24P4C12 fusion protein or analog etc.) in a vaccine preparation to ahuman or another mammal. Typically, such vaccine preparations furthercontain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or auniversal helper epitope such as a PADRET peptide (Epimmune Inc., SanDiego, Calif.; see, e.g., Alexander et al., J. Immunol. 2000 164(3);164(3): 1625-1633; Alexander et al., Immunity 1994 1(9): 751-761 andAlexander et al., Immunol. Res. 1998 18(2): 79-92). An alternativemethod comprises generating an immune response in an individual againsta 24P4C12 immunogen by: administering in vivo to muscle or skin of theindividual's body a DNA molecule that comprises a DNA sequence thatencodes a 24P4C12 immunogen, the DNA sequence operatively linked toregulatory sequences which control the expression of the DNA sequence;wherein the DNA molecule is taken up by cells, the DNA sequence isexpressed in the cells and an immune response is generated against theimmunogen (see, e.g., U.S. Pat. No. 5,962,428). Optionally a geneticvaccine facilitator such as anionic lipids; saponins; lectins;estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; andurea is also administered. In addition, an antiidiotypic antibody can beadministered that mimics 24P4C12, in order to generate a response to thetarget antigen.

Nucleic Acid Vaccines:

Vaccine compositions of the invention include nucleic acid-mediatedmodalities. DNA or RNA that encode protein(s) of the invention can beadministered to a patient. Genetic immunization methods can be employedto generate prophylactic or therapeutic humoral and cellular immuneresponses directed against cancer cells expressing 24P4C12. Constructscomprising DNA encoding a 24P4C12-related protein/immunogen andappropriate regulatory sequences can be injected directly into muscle orskin of an individual, such that the cells of the muscle or skin take-upthe construct and express the encoded 24P4C12 protein/immunogen.Alternatively, a vaccine comprises a 24P4C12-related protein. Expressionof the 24P4C12-related protein immunogen results in the generation ofprophylactic or therapeutic humoral and cellular immunity against cellsthat bear a 24P4C12 protein. Various prophylactic and therapeuticgenetic immunization techniques known in the art can be used (forreview, see information and references published at Internet addressgenweb.com). Nucleic acid-based delivery is described, for instance, inWolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos.5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO98/04720. Examples of DNA-based delivery technologies include “nakedDNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery,cationic lipid complexes, and particle-mediated (“gene gun”) orpressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).

For therapeutic or prophylactic immunization purposes, proteins of theinvention can be expressed via viral or bacterial vectors. Various viralgene delivery systems that can be used in the practice of the inventioninclude, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol.8:658-663; Tsang et al. J. Natl. Cancer Inst. 87:982-990 (1995)).Non-viral delivery systems can also be employed by introducing naked DNAencoding a 24P4C12-related protein into the patient (e.g.,intramuscularly or intradermally) to induce an anti-tumor response.

Vaccinia virus is used, for example, as a vector to express nucleotidesequences that encode the peptides of the invention. Upon introductioninto a host, the recombinant vaccinia virus expresses the proteinimmunogenic peptide, and thereby elicits a host immune response.Vaccinia vectors and methods useful in immunization protocols aredescribed in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG(Bacille Calmette Guerin). BCG vectors are described in Stover et al.,Nature 351:456-460 (1991). A wide variety of other vectors useful fortherapeutic administration or immunization of the peptides of theinvention, e.g. adeno and adeno-associated virus vectors, retroviralvectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, andthe like, will be apparent to those skilled in the art from thedescription herein.

Thus, gene delivery systems are used to deliver a 24P4C12-relatednucleic acid molecule. In one embodiment, the full-length human 24P4C12cDNA is employed. In another embodiment, 24P4C12 nucleic acid moleculesencoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopesare employed.

Ex Vivo Vaccines

Various ex vivo strategies can also be employed to generate an immuneresponse. One approach involves the use of antigen presenting cells(APCs) such as dendritic cells (DC) to present 24P4C12 antigen to apatients immune system. Dendritic cells express MHC class I and IImolecules, B7 co-stimulator, and IL-12, and are thus highly specializedantigen presenting cells. In prostate cancer, autologous dendritic cellspulsed with peptides of the prostate-specific membrane antigen (PSMA)are being used in a Phase I clinical trial to stimulate prostate cancerpatients' immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphyet al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used topresent 24P4C12 peptides to T cells in the context of MHC class I or IImolecules. In one embodiment, autologous dendritic cells are pulsed with24P4C12 peptides capable of binding to MHC class I and/or class IImolecules. In another embodiment, dendritic cells are pulsed with thecomplete 24P4C12 protein. Yet another embodiment involves engineeringthe overexpression of a 24P4C12 gene in dendritic cells using variousimplementing vectors known in the art, such as adenovirus (Arthur etal., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al.,1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNAtransfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), ortumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med.186:1177-1182). Cells that express 24P4C12 can also be engineered toexpress immune modulators, such as GM-CSF, and used as immunizingagents.

X.B.) 24P4C12 as a Target for Antibody-Based Therapy

24P4C12 is an attractive target for antibody-based therapeuticstrategies. A number of antibody strategies are known in the art fortargeting both extracellular and intracellular molecules (see, e.g.,complement and ADCC mediated killing as well as the use of intrabodies).Because 24P4C12 is expressed by cancer cells of various lineagesrelative to corresponding normal cells, systemic administration of24P4C12-immunoreactive compositions are prepared that exhibit excellentsensitivity without toxic, non-specific and/or non-target effects causedby binding of the immunoreactive composition to non-target organs andtissues. Antibodies specifically reactive with domains of 24P4C12 areuseful to treat 24P4C12-expressing cancers systemically, either asconjugates with a toxin or therapeutic agent, or as naked antibodiescapable of inhibiting cell proliferation or function.

24P4C12 antibodies can be introduced into a patient such that theantibody binds to 24P4C12 and modulates a function, such as aninteraction with a binding partner, and consequently mediatesdestruction of the tumor cells and/or inhibits the growth of the tumorcells. Mechanisms by which such antibodies exert a therapeutic effectcan include complement-mediated cytolysis, antibody-dependent cellularcytotoxicity, modulation of the physiological function of 24P4C12,inhibition of ligand binding or signal transduction pathways, modulationof tumor cell differentiation, alteration of tumor angiogenesis factorprofiles, and/or apoptosis.

Those skilled in the art understand that antibodies can be used tospecifically target and bind immunogenic molecules such as animmunogenic region of a 24P4C12 sequence shown in FIG. 2 or FIG. 3. Inaddition, skilled artisans understand that it is routine to conjugateantibodies to cytotoxic agents (see, e.g., Slevers et al. Blood 93:113678-3684 (Jun. 1, 1999)). When cytotoxic and/or therapeutic agents aredelivered directly to cells, such as by conjugating them to antibodiesspecific for a molecule expressed by that cell (e.g. 24P4C12), thecytotoxic agent will exert its known biological effect (i.e.cytotoxicity) on those cells.

A wide variety of compositions and methods for using antibody-cytotoxicagent conjugates to kill cells are known in the art. In the context ofcancers, typical methods entail administering to an animal having atumor a biologically effective amount of a conjugate comprising aselected cytotoxic and/or therapeutic agent linked to a targeting agent(e.g. an anti-24P4C12 antibody) that binds to a marker (e.g. 24P4C12)expressed, accessible to binding or localized on the cell surfaces. Atypical embodiment is a method of delivering a cytotoxic and/ortherapeutic agent to a cell expressing 24P4C12, comprising conjugatingthe cytotoxic agent to an antibody that immunospecifically binds to a24P4C12 epitope, and, exposing the cell to the antibody-agent conjugate.Another illustrative embodiment is a method of treating an individualsuspected of suffering from metastasized cancer, comprising a step ofadministering parenterally to said individual a pharmaceuticalcomposition comprising a therapeutically effective amount of an antibodyconjugated to a cytotoxic and/or therapeutic agent.

Cancer immunotherapy using anti-24P4C12 antibodies can be done inaccordance with various approaches that have been successfully employedin the treatment of other types of cancer, including but not limited tocolon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138),multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari etal., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk etaaL, 1992,Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J.Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al.,1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al., 1994,Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res.55:4398-4403), and breast cancer (Shepard et al., 1991, J, Clin.Immunol. 11:117-127). Some therapeutic approaches involve conjugation ofnaked antibody to a toxin or radioisotope, such as the conjugation ofY⁹¹ or I¹³¹ to anti-CD20 antibodies (e.g., Zevalin™, IDECPharmaceuticals Corp. or Bexxar™, Coulter Pharmaceuticals), while othersinvolve co-administration of antibodies and other therapeutic agents,such as Herceptinm (trastuzumab) with paclitaxel (Genentech, Inc.). Theantibodies can be conjugated to a therapeutic agent. To treat prostatecancer, for example, 24P4C12 antibodies can be administered inconjunction with radiation, chemotherapy or hormone ablation. Also,antibodies can be conjugated to a toxin such as calicheamicin (e.g.,Mylotarg™, Wyeth-Ayerst, Madison, N.J., a recombinant humanized IgG₄kappa antibody conjugated to antitumor antibiotic calicheamicin) or amaytansinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP, platform,ImmunoGen, Cambridge, Mass., also see e.g., U.S. Pat. No. 5,416,064).

Although 24P4C12 antibody therapy is useful for all stages of cancer,antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well. Fan et al. (Cancer Res.53:4637-4642, 1993), Prewett et al. (International J. of Onco.9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580, 1991)describe the use of various antibodies together with chemotherapeuticagents.

Although 24P4C12 antibody therapy is useful for all stages of cancer,antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well.

Cancer patients can be evaluated for the presence and level of 24P4C12expression, preferably using immunohistochemical assessments of tumortissue, quantitative 24P4C12 imaging, or other techniques that reliablyindicate the presence and degree of 24P4C12 expression.Immunohistochemical analysis of tumor biopsies or surgical specimens ispreferred for this purpose. Methods for immunohistochemical analysis oftumor tissues are well known in the art.

Anti-24P4C12 monoclonal antibodies that treat prostate and other cancersinclude those that initiate a potent immune response against the tumoror those that are directly cytotoxic. In this regard, anti-24P4C12monoclonal antibodies (mAbs) can elicit tumor cell lysis by eithercomplement-mediated or antibody-dependent cell cytotoxicity (ADCC)mechanisms, both of which require an intact Fc portion of theimmunoglobulin molecule for interaction with effector cell Fc receptorsites on complement proteins. In addition, anti-24P4C12 mAbs that exerta direct biological effect on tumor growth are useful to treat cancersthat express 24P4C12. Mechanisms by which directly cytotoxic mAbs actinclude: inhibition of cell growth, modulation of cellulardifferentiation, modulation of tumor angiogenesis factor profiles, andthe induction of apoptosis. The mechanism(s) by which a particularanti-24P4C12 mAb exerts an anti-tumor effect is evaluated using anynumber of in vitro assays that evaluate cell death such as ADCC, ADMMC,complement-mediated cell lysis, and so forth, as is generally known inthe art.

In some patients, the use of murine or other non-human monoclonalantibodies, or human/mouse chimeric mAbs can induce moderate to strongimmune responses against the non-human antibody. This can result inclearance of the antibody from circulation and reduced efficacy. In themost severe cases, such an immune response can lead to the extensiveformation of immune complexes which, potentially, can cause renalfailure. Accordingly, preferred monoclonal antibodies used in thetherapeutic methods of the invention are those that are either fullyhuman or humanized and that bind specifically to the target 24P4C12antigen with high affinity but exhibit low or no antigenicity in thepatient.

Therapeutic methods of the invention contemplate the administration ofsingle anti-24P4C12 mAbs as well as combinations, or cocktails, ofdifferent mAbs. Such mAb cocktails can have certain advantages inasmuchas they contain mAbs that target different epitopes, exploit differenteffector mechanisms or combine directly cytotoxic mAbs with mAbs thatrely on immune effector functionality. Such mAbs in combination canexhibit synergistic therapeutic effects. In addition, anti-24P4C12 mAbscan be administered concomitantly with other therapeutic modalities,including but not limited to various chemotherapeutic agents,androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery orradiation. The anti-24P4C12 mAbs are administered in their “naked” orunconjugated form, or can have a therapeutic agent(s) conjugated tothem.

Anti-24P4C12 antibody formulations are administered via any routecapable of delivering the antibodies to a tumor cell. Routes ofadministration include, but are not limited to, intravenous,intraperitoneal, intramuscular, intratumor, intradermal, and the like.Treatment generally involves repeated administration of the anti-24P4C12antibody preparation, via an acceptable route of administration such asintravenous injection (IV), typically at a dose in the range of about0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, or 25 mg/kg body weight. In general, doses in the range of10-1000 mg mAb per week are effective and well tolerated.

Based on clinical experience with the Herceptin™ mAb in the treatment ofmetastatic breast cancer, an initial loading dose of approximately 4mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kgIV of the anti-24P4C12 mAb preparation represents an acceptable dosingregimen. Preferably, the initial loading dose is administered as a90-minute or longer infusion. The periodic maintenance dose isadministered as a 30 minute or longer infusion, provided the initialdose was well tolerated. As appreciated by those of skill in the art,various factors can influence the ideal dose regimen in a particularcase. Such factors include, for example, the binding affinity and halflife of the Ab or mAbs used, the degree of 24P4C12 expression in thepatient, the extent of circulating shed 24P4C12 antigen, the desiredsteady-state antibody concentration level, frequency of treatment, andthe influence of chemotherapeutic or other agents used in combinationwith the treatment method of the invention, as well as the health statusof a particular patient.

Optionally, patients should be evaluated for the levels of 24P4C12 in agiven sample (e.g. the levels of circulating 24P4C12 antigen and/or24P4C12 expressing cells) in order to assist in the determination of themost effective dosing regimen, etc. Such evaluations are also used formonitoring purposes throughout therapy, and are useful to gaugetherapeutic success in combination with the evaluation of otherparameters (for example, urine cytology and/or ImmunoCyt levels inbladder cancer therapy, or by analogy, serum PSA levels in prostatecancer therapy).

Anti-idiotypic anti-24P4C12 antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga 24P4C12-related protein. In particular, the generation ofanti-idiotypic antibodies is well known in the art; this methodology canreadily be adapted to generate anti-idiotypic anti-24P4C12 antibodiesthat mimic an epitope on a 24P4C12-related protein (see, for example,Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J. Clin.Invest. 96:334-342; Herlyn et al., 1996, Cancer Immunol. Immunother.43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccinestrategies.

X.C.) 24P4C12 as a Target for Cellular Immune Responses

Vaccines and methods of preparing vaccines that contain animmunogenically effective amount of one or more HLA-binding peptides asdescribed herein are further embodiments of the invention. Furthermore,vaccines in accordance with the invention encompass compositions of oneor more of the claimed peptides. A peptide can be present in a vaccineindividually. Alternatively, the peptide can exist as a homopolymercomprising multiple copies of the same peptide, or as a heteropolymer ofvarious peptides. Polymers have the advantage of increased immunologicalreaction and, where different peptide epitopes are used to make up thepolymer, the additional ability to induce antibodies and/or CTLs thatreact with different antigenic determinants of the pathogenic organismor tumor-related peptide targeted for an immune response. Thecomposition can be a naturally occurring region of an antigen or can beprepared, e.g., recombinantly or by chemical synthesis.

Carriers that can be used with vaccines of the invention are well knownin the art, and include, e.g., thyroglobulin, albumins such as humanserum albumin, tetanus toxoid, polyamino acids such as poly L-lysine,poly L-glutamic acid, influenza, hepatitis B virus core protein, and thelike. The vaccines can contain a physiologically tolerable (i.e.,acceptable) diluent such as water, or saline, preferably phosphatebuffered saline. The vaccines also typically include an adjuvant.Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate,aluminum hydroxide, or alum are examples of materials well known in theart. Additionally, as disclosed herein, CTL responses can be primed byconjugating peptides of the invention to lipids, such astripalmitoyl-S-glycerylcysteinlyseryl-serine (P₃CSS). Moreover, anadjuvant such as a syntheticcytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotideshas been found to increase CTL responses 10- to 100-fold. (see, e.g.Davila and Celis, J. Immunol. 165:539-547 (2000))

Upon immunization with a peptide composition in accordance with theinvention, via injection, aerosol, oral, transdermal, transmucosal,intrapleural, intrathecal, or other suitable routes, the immune systemof the host responds to the vaccine by producing large amounts of CTLsand/or HTLs specific for the desired antigen. Consequently, the hostbecomes at least partially immune to later development of cells thatexpress or overexpress 24P4C12 antigen, or derives at least sometherapeutic benefit when the antigen was tumor-associated.

In some embodiments, it may be desirable to combine the class I peptidecomponents with components that induce or facilitate neutralizingantibody and or helper T cell responses directed to the target antigen.A preferred embodiment of such a composition comprises class I and classII epitopes in accordance with the invention. An alternative embodimentof such a composition comprises a class I and/or class II epitope inaccordance with the invention, along with a cross reactive HTL epitopesuch as PADRE™ (Epimmune, San Diego, Calif.) molecule (described e.g.,in U.S. Pat. No. 5,736,142).

A vaccine of the invention can also include antigen-presenting cells(APC), such as dendritic cells (DC), as a vehicle to present peptides ofthe invention. Vaccine compositions can be created in vitro, followingdendritic cell mobilization and harvesting, whereby loading of dendriticcells occurs in vitro. For example, dendritic cells are transfected,e.g., with a minigene in accordance with the invention, or are pulsedwith peptides. The dendritic cell can then be administered to a patientto elicit immune responses in vivo. Vaccine compositions, either DNA- orpeptide-based, can also be administered in vivo in combination withdendritic cell mobilization whereby loading of dendritic cells occurs invivo.

Preferably, the following principles are utilized when selecting anarray of epitopes for inclusion in a polyepitopic composition for use ina vaccine, or for selecting discrete epitopes to be included in avaccine and/or to be encoded by nucleic acids such as a minigene. It ispreferred that each of the following principles be balanced in order tomake the selection. The multiple epitopes to be incorporated in a givenvaccine composition may be, but need not be, contiguous in sequence inthe native antigen from which the epitopes are derived.

1.) Epitopes are selected which, upon administration, mimic immuneresponses that have been observed to be correlated with tumor clearance.For HLA Class I this includes 3-4 epitopes that come from at least onetumor associated antigen (TAA). For HLA Class II a similar rationale isemployed; again 3-4 epitopes are selected from at least one TAA (see,e.g., Rosenberg et al., Science 278:1447-1450). Epitopes from one TAAmay be used in combination with epitopes from one or more additionalTAAs to produce a vaccine that targets tumors with varying expressionpatterns of frequently-expressed TMs.

2.) Epitopes are selected that have the requisite binding affinityestablished to be correlated with immunogenicity: for HLA Class I anIC₅₀ of 500 nM or less, often 200 nM or less; and for Class II an IC₅₀of 1000 nM or less.

3.) Sufficient supermotif bearing-peptides, or a sufficient array ofallele-specific motif-bearing peptides, are selected to give broadpopulation coverage. For example, it is preferable to have at least 80%population coverage. A Monte Carlo analysis, a statistical evaluationknown in the art, can be employed to assess the breadth, or redundancyof, population coverage.

4.) When selecting epitopes from cancer-related antigens it is oftenuseful to select analogs because the patient may have developedtolerance to the native epitope.

5.) Of particular relevance are epitopes referred to as “nestedepitopes.” Nested epitopes occur where at least two epitopes overlap ina given peptide sequence. A nested peptide sequence can comprise B cell,HLA class I and/or HLA class II epitopes. When providing nestedepitopes, a general objective is to provide the greatest number ofepitopes per sequence. Thus, an aspect is to avoid providing a peptidethat is any longer than the amino terminus of the amino terminal epitopeand the carboxyl terminus of the carboxyl terminal epitope in thepeptide. When providing a multi-epitopic sequence, such as a sequencecomprising nested epitopes, it is generally important to screen thesequence in order to insure that it does not have pathological or otherdeleterious biological properties.

6.) If a polyepitopic protein is created, or when creating a minigene,an objective is to generate the smallest peptide that encompasses theepitopes of interest. This principle is similar, if not the same as thatemployed when selecting a peptide comprising nested epitopes. However,with an artificial polyepitopic peptide, the size minimization objectiveis balanced against the need to integrate any spacer sequences betweenepitopes in the polyepitopic protein. Spacer amino acid residues can,for example, be introduced to avoid junctional epitopes (an epitoperecognized by the immune system, not present in the target antigen, andonly created by the man-made juxtaposition of epitopes), or tofacilitate cleavage between epitopes and thereby enhance epitopepresentation. Junctional epitopes are generally to be avoided becausethe recipient may generate an immune response to that non-nativeepitope. Of particular concern is a junctional epitope that is a“dominant epitope.” A dominant epitope may lead to such a zealousresponse that immune responses to other epitopes are diminished orsuppressed.

7.) Where the sequences of multiple variants of the same target proteinare present, potential peptide epitopes can also be selected on thebasis of their conservancy. For example, a criterion for conservancy maydefine that the entire sequence of an HLA class I binding peptide or theentire 9-mer core of a class II binding peptide be conserved in adesignated percentage of the sequences evaluated for a specific proteinantigen.

X.C.1. Minigene Vaccines

A number of different approaches are available which allow simultaneousdelivery of multiple epitopes. Nucleic acids encoding the peptides ofthe invention are a particularly useful embodiment of the invention.Epitopes for inclusion in a minigene are preferably selected accordingto the guidelines set forth in the previous section. A preferred meansof administering nucleic acids encoding the peptides of the inventionuses minigene constructs encoding a peptide comprising one or multipleepitopes of the invention.

The use of multi-epitope minigenes is described below and in, Ishioka etal., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J.Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996;Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine16:426, 1998. For example, a multi-epitope DNA plasmid encodingsupermotif- and/or motif-bearing epitopes derived 24P4C12, the PADRE®universal helper T cell epitope or multiple HTL epitopes from 24P4C12(see e.g., Tables VIII-XXI and XXII to XLIX), and an endoplasmicreticulum-translocating signal sequence can be engineered. A vaccine mayalso comprise epitopes that are derived from other TAAs.

The immunogenicity of a multi-epitopic minigene can be confirmed intransgenic mice to evaluate the magnitude of CTL induction responsesagainst the epitopes tested. Further, the immunogenicity of DNA-encodedepitopes in vivo can be correlated with the in vitro responses ofspecific CTL lines against target cells transfected with the DNAplasmid. Thus, these experiments can show that the minigene serves toboth: 1.) generate a CTL response and 2.) that the induced CTLsrecognized cells expressing the encoded epitopes.

For example, to create a DNA sequence encoding the selected epitopes(minigene) for expression in human cells, the amino acid sequences ofthe epitopes may be reverse translated. A human codon usage table can beused to guide the codon choice for each amino acid. Theseepitope-encoding DNA sequences may be directly adjoined, so that whentranslated, a continuous polypeptide sequence is created. To optimizeexpression and/or immunogenicity, additional elements can beincorporated into the minigene design. Examples of amino acid sequencesthat can be reverse translated and included in the minigene sequenceinclude: HLA class I epitopes, HLA class II epitopes, antibody epitopes,a ubiquitination signal sequence, and/or an endoplasmic reticulumtargeting signal. In addition, HLA presentation of CTL and HTL epitopesmay be improved by including synthetic (e.g. poly-alanine) ornaturally-occurring flanking sequences adjacent to the CTL or HTLepitopes; these larger peptides comprising the epitope(s) are within thescope of the invention.

The minigene sequence may be converted to DNA by assemblingoligonucleotides that encode the plus and minus strands of the minigene.Overlapping oligonucleotides (30-100 bases long) may be synthesized,phosphorylated, purified and annealed under appropriate conditions usingwell known techniques. The ends of the oligonucleotides can be joined,for example, using T4 DNA ligase. This synthetic minigene, encoding theepitope polypeptide, can then be cloned into a desired expressionvector.

Standard regulatory sequences well known to those of skill in the artare preferably included in the vector to ensure expression in the targetcells. Several vector elements are desirable: a promoter with adown-stream cloning site for minigene insertion; a polyadenylationsignal for efficient transcription termination; an E. coli origin ofreplication; and an E. coli selectable marker (e.g. ampicillin orkanamycin resistance). Numerous promoters can be used for this purpose,e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat.Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigeneexpression and immunogenicity. In some cases, introns are required forefficient gene expression, and one or more synthetic ornaturally-occurring introns could be incorporated into the transcribedregion of the minigene. The inclusion of mRNA stabilization sequencesand sequences for replication in mammalian cells may also be consideredfor increasing minigene expression.

Once an expression vector is selected, the minigene is cloned into thepolylinker region downstream of the promoter. This plasmid istransformed into an appropriate E. coli strain, and DNA is preparedusing standard techniques. The orientation and DNA sequence of theminigene, as well as all other elements included in the vector, areconfirmed using restriction mapping and DNA sequence analysis. Bacterialcells harboring the correct plasmid can be stored as a master cell bankand a working cell bank.

In addition, immunostimulatory sequences (ISSs or CpGs) appear to play arole in the immunogenicity of DNA vaccines. These sequences may beincluded in the vector, outside the minigene coding sequence, if desiredto enhance immunogenicity.

In some embodiments, a bi-cistronic expression vector which allowsproduction of both the minigene-encoded epitopes and a second protein(included to enhance or decrease immunogenicity) can be used. Examplesof proteins or polypeptides that could beneficially enhance the immuneresponse if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF),cytokine-inducing molecules (e.g., LelF), costimulatory molecules, orfor HTL responses, pan-DR binding proteins (PADRE™, Epimmune, San Diego,Calif.). Helper (HTL) epitopes can be joined to intracellular targetingsignals and expressed separately from expressed CTL epitopes; thisallows direction of the HTL epitopes to a cell compartment differentthan that of the CTL epitopes. If required, this could facilitate moreefficient entry of HTL epitopes into the HLA class II pathway, therebyimproving HTL induction. In contrast to HTL or CTL induction,specifically decreasing the immune response by co-expression ofimmunosuppressive molecules (e.g. TGF-β) may be beneficial in certaindiseases.

Therapeutic quantities of plasmid DNA can be produced for example, byfermentation in E. coli, followed by purification. Aliquots from theworking cell bank are used to inoculate growth medium, and grown tosaturation in shaker flasks or a bioreactor according to well-knowntechniques. Plasmid DNA can be purified using standard bioseparationtechnologies such as solid phase anion-exchange resins supplied byQIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can beisolated from the open circular and linear forms using gelelectrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety offormulations. The simplest of these is reconstitution of lyophilized DNAin sterile phosphate-buffer saline (PBS). This approach, known as “nakedDNA,” is currently being used for intramuscular (IM) administration inclinical trials. To maximize the immunotherapeutic effects of minigeneDNA vaccines, an alternative method for formulating purified plasmid DNAmay be desirable. A variety of methods have been described, and newtechniques may become available. Cationic lipids, glycolipids, andfusogenic liposomes can also be used in the formulation (see, e.g., asdescribed by WO 93/24640; Mannino & Gould-Fogerite, Bio Techniques 6(7):682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Feigner, et al.,Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In addition, peptides andcompounds referred to collectively as protective, interactive,non-condensing compounds (PINC) could also be complexed to purifiedplasmid DNA to influence variables such as stability, intramusculardispersion, or trafficking to specific organs or cell types.

Target cell sensitization can be used as a functional assay forexpression and HLA class I presentation of minigene-encoded CTLepitopes. For example, the plasmid DNA is introduced into a mammaliancell line that is suitable as a target for standard CTL chromium releaseassays. The transfection method used will be dependent on the finalformulation. Electroporation can be used for “naked” DNA, whereascationic lipids allow direct in vitro transfection. A plasmid expressinggreen fluorescent protein (GFP) can be co-transfected to allowenrichment of transfected cells using fluorescence activated cellsorting (FACS). These cells are then chromium-51 (⁵¹Cr) labeled and usedas target cells for epitope-specific CTL lines; cytolysis, detected by⁵¹Cr release, indicates both production of, and HLA presentation of,minigene-encoded CTL epitopes. Expression of HTL epitopes may beevaluated in an analogous manner using assays to assess HTL activity.

In vivo immunogenicity is a second approach for functional testing ofminigene DNA formulations. Transgenic mice expressing appropriate humanHLA proteins are immunized with the DNA product. The dose and route ofadministration are formulation dependent (e.g., IM for DNA in PBS,intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days afterimmunization, splenocytes are harvested and restimulated for one week inthe presence of peptides encoding each epitope being tested. Thereafter,for CTL effector cells, assays are conducted for cytolysis ofpeptide-loaded, ⁵¹Cr-labeled target cells using standard techniques.Lysis of target cells that were sensitized by HLA loaded with peptideepitopes, corresponding to minigene-encoded epitopes, demonstrates DNAvaccine function for in vivo induction of CTLs. Immunogenicity of HTLepitopes is confirmed in transgenic mice in an analogous manner.

Alternatively, the nucleic acids can be administered using ballisticdelivery as described, for instance, in U.S. Pat. No. 5,204,253. Usingthis technique, particles comprised solely of DNA are administered. In afurther alternative embodiment, DNA can be adhered to particles, such asgold particles.

Minigenes can also be delivered using other bacterial or viral deliverysystems well known in the art, e.g., an expression construct encodingepitopes of the invention can be incorporated into a viral vector suchas vaccinia.

X.C.2. Combinations of CTL Peptides with Helper Peptides

Vaccine compositions comprising CTL peptides of the invention can bemodified, e.g., analoged, to provide desired attributes, such asimproved serum half life, broadened population coverage or enhancedimmunogenicity.

For instance, the ability of a peptide to induce CTL activity can beenhanced by linking the peptide to a sequence which contains at leastone epitope that is capable of inducing a T helper cell response.Although a CTL peptide can be directly linked to a T helper peptide,often CTL epitope/HTL epitope conjugates are linked by a spacermolecule. The spacer is typically comprised of relatively small, neutralmolecules, such as amino acids or amino acid mimetics, which aresubstantially uncharged under physiological conditions. The spacers aretypically selected from, e.g., Ala, Gly, or other neutral spacers ofnonpolar amino acids or neutral polar amino acids. It will be understoodthat the optionally present spacer need not be comprised of the sameresidues and thus may be a hetero- or homo-oligomer. When present, thespacer will usually be at least one or two residues, more usually threeto six residues and sometimes 10 or more residues. The CTL peptideepitope can be linked to the T helper peptide epitope either directly orvia a spacer either at the amino or carboxy terminus of the CTL peptide.The amino terminus of either the immunogenic peptide or the T helperpeptide may be acylated.

In certain embodiments, the T helper peptide is one that is recognizedby T helper cells present in a majority of a genetically diversepopulation. This can be accomplished by selecting peptides that bind tomany, most, or all of the HLA class II molecules. Examples of such aminoacid bind many HLA Class II molecules include sequences from antigenssuch as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO:29), Plasmodium falciparum circumsporozoite (CS) protein at positions378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 30), and Streptococcus 18 kDprotein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: 31). Otherexamples include peptides bearing a DR 1-4-7 supermotif, or either ofthe DR3 motifs.

Alternatively, it is possible to prepare synthetic peptides capable ofstimulating T helper lymphocytes, in a loosely HLA-restricted fashion,using amino acid sequences not found in nature (see, e.g., PCTpublication WO 95/07707). These synthetic compounds calledPan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego,Calif.) are designed, most preferably, to bind most HLA-DR (human HLAclass II) molecules. For instance, a pan-DR-binding epitope peptidehaving the formula: AKXVAAWTLKAAA (SEQ ID NO: 32), where “X” is eithercyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanineor L-alanine, has been found to bind to most HLA-DR alleles, and tostimulate the response of T helper lymphocytes from most individuals,regardless of their HLA type. An alternative of a pan-DR binding epitopecomprises all “L” natural amino acids and can be provided in the form ofnucleic acids that encode the epitope.

HTL peptide epitopes can also be modified to alter their biologicalproperties. For example, they can be modified to include D-amino acidsto increase their resistance to proteases and thus extend their serumhalf life, or they can be conjugated to other molecules such as lipids,proteins, carbohydrates, and the like to increase their biologicalactivity. For example, a T helper peptide can be conjugated to one ormore palmitic acid chains at either the amino or carboxyl termini.

X.C.3. Combinations of CTL Peptides with T Cell Priming Agents

In some embodiments it may be desirable to include in the pharmaceuticalcompositions of the invention at least one component which primes Blymphocytes or T lymphocytes. Lipids have been identified as agentscapable of priming CTL in vivo. For example, palmitic acid residues canbe attached to the ε- and α-amino groups of a lysine residue and thenlinked, e.g., via one or more linking residues such as Gly, Gly-Gly-,Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidatedpeptide can then be administered either directly in a micelle orparticle, incorporated into a liposome, or emulsified in an adjuvant,e.g., incomplete Freund's adjuvant. In a preferred embodiment, aparticularly effective immunogenic composition comprises palmitic acidattached to ε- and α-amino groups of Lys, which is attached via linkage,e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine(P₃CSS) can be used to prime virus specific CTL when covalently attachedto an appropriate peptide (see, e.g., Deres, et al., Nature 342:561,1989). Peptides of the invention can be coupled to P₃CSS, for example,and the lipopeptide administered to an individual to prime specificallyan immune response to the target antigen. Moreover, because theinduction of neutralizing antibodies can also be primed withP₃CSS-conjugated epitopes, two such compositions can be combined to moreeffectively elicit both humoral and cell-mediated responses.

X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTLPeptides

An embodiment of a vaccine composition in accordance with the inventioncomprises ex vivo administration of a cocktail of epitope-bearingpeptides to PBMC, or isolated DC therefrom, from the patient's blood. Apharmaceutical to facilitate harvesting of DC can be used, such asProgenipoietin™ (Pharmacia-Monsanto, St. Louis, Mo.) or GM-CSF/IL-4.After pulsing the DC with peptides and prior to reinfusion intopatients, the DC are washed to remove unbound peptides. In thisembodiment, a vaccine comprises peptide-pulsed DCs which present thepulsed peptide epitopes complexed with HLA molecules on their surfaces.

The DC can be pulsed ex vivo with a cocktail of peptides, some of whichstimulate CTL responses to 24P4C12. Optionally, a helper T cell (HTL)peptide, such as a natural or artificial loosely restricted HLA Class IIpeptide, can be included to facilitate the CTL response. Thus, a vaccinein accordance with the invention is used to treat a cancer whichexpresses or overexpresses 24P4C12.

X.D. Adoptive Immunotherapy

Antigenic 24P4C12-related peptides are used to elicit a CTL and/or HTLresponse ex vivo, as well. The resulting CTL or HTL cells, can be usedto treat tumors in patients that do not respond to other conventionalforms of therapy, or will not respond to a therapeutic vaccine peptideor nucleic acid in accordance with the invention. Ex vivo CTL or HTLresponses to a particular antigen are induced by incubating in tissueculture the patient's, or genetically compatible, CTL or HTL precursorcells together with a source of antigen-presenting cells (APC), such asdendritic cells, and the appropriate immunogenic peptide. After anappropriate incubation time (typically about 7-28 days), in which theprecursor cells are activated and expanded into effector cells, thecells are infused back into the patient, where they will destroy (CTL)or facilitate destruction (HTL) of their specific target cell (e.g., atumor cell). Transfected dendritic cells may also be used as antigenpresenting cells.

X.E. Administration of Vaccines for Therapeutic or Prophylactic Purposes

Pharmaceutical and vaccine compositions of the invention are typicallyused to treat and/or prevent a cancer that expresses or overexpresses24P4C12. In therapeutic applications, peptide and/or nucleic acidcompositions are administered to a patient in an amount sufficient toelicit an effective B cell, CTL and/or HTL response to the antigen andto cure or at least partially arrest or slow symptoms and/orcomplications. An amount adequate to accomplish this is defined as“therapeutically effective dose.” Amounts effective for this use willdepend on, e.g., the particular composition administered, the manner ofadministration, the stage and severity of the disease being treated, theweight and general state of health of the patient, and the judgment ofthe prescribing physician.

For pharmaceutical compositions, the immunogenic peptides of theinvention, or DNA encoding them, are generally administered to anindividual already bearing a tumor that expresses 24P4C12. The peptidesor DNA encoding them can be administered individually or as fusions ofone or more peptide sequences. Patients can be treated with theimmunogenic peptides separately or in conjunction with other treatments,such as surgery, as appropriate.

For therapeutic use, administration should generally begin at the firstdiagnosis of 24P4C12-associated cancer. This is followed by boostingdoses until at least symptoms are substantially abated and for a periodthereafter. The embodiment of the vaccine composition (i.e., including,but not limited to embodiments such as peptide cocktails, polyepitopicpolypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells)delivered to the patient may vary according to the stage of the diseaseor the patient's health status. For example, in a patient with a tumorthat expresses 24P4C12, a vaccine comprising 24P4C12-specific CTL may bemore efficacious in killing tumor cells in patient with advanced diseasethan alternative embodiments.

It is generally important to provide an amount of the peptide epitopedelivered by a mode of administration sufficient to stimulateeffectively a cytotoxic T cell response; compositions which stimulatehelper T cell responses can also be given in accordance with thisembodiment of the invention.

The dosage for an initial therapeutic immunization generally occurs in aunit dosage range where the lower value is about 1, 5, 50, 500, or 1,000μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg.Dosage values for a human typically range from about 500 μg to about50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0μg to about 50,000 μg of peptide pursuant to a boosting regimen overweeks to months may be administered depending upon the patient'sresponse and condition as determined by measuring the specific activityof CTL and HTL obtained from the patient's blood. Administration shouldcontinue until at least clinical symptoms or laboratory tests indicatethat the neoplasia, has been eliminated or reduced and for a periodthereafter. The dosages, routes of administration, and dose schedulesare adjusted in accordance with methodologies known in the art.

In certain embodiments, the peptides and compositions of the presentinvention are employed in serious disease states, that is,life-threatening or potentially life threatening situations. In suchcases, as a result of the minimal amounts of extraneous substances andthe relative nontoxic nature of the peptides in preferred compositionsof the invention, it is possible and may be felt desirable by thetreating physician to administer substantial excesses of these peptidecompositions relative to these stated dosage amounts.

The vaccine compositions of the invention can also be used purely asprophylactic agents. Generally the dosage for an initial prophylacticimmunization generally occurs in a unit dosage range where the lowervalue is about 1, 5, 50, 500, or 1000 μg and the higher value is about10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a humantypically range from about 500 μg to about 50,000 μg per 70 kilogrampatient. This is followed by boosting dosages of between about 1.0 μg toabout 50,000 μg of peptide administered at defined intervals from aboutfour weeks to six months after the initial administration of vaccine.The immunogenicity of the vaccine can be assessed by measuring thespecific activity of CTL and HTL obtained from a sample of the patient'sblood.

The pharmaceutical compositions for therapeutic treatment are intendedfor parenteral, topical, oral, nasal, intrathecal, or local (e.g. as acream or topical ointment) administration. Preferably, thepharmaceutical compositions are administered parentally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. Thus,the invention provides compositions for parenteral administration whichcomprise a solution of the immunogenic peptides dissolved or suspendedin an acceptable carrier, preferably an aqueous carrier.

A variety of aqueous carriers may be used, e.g., water, buffered water,0.8% saline, 0.3% glycine, hyaluronic acid and the like. Thesecompositions may be sterilized by conventional, well-known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration.

The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH-adjusting and buffering agents, tonicity adjusting agents, wettingagents, preservatives, and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, sorbitanmonolaurate, triethanolamine oleate, etc.

The concentration of peptides of the invention in the pharmaceuticalformulations can vary widely, i.e., from less than about 0.1%, usuallyat or at least about 2% to as much as 20% to 50% or more by weight, andwill be selected primarily by fluid volumes, viscosities, etc., inaccordance with the particular mode of administration selected.

A human unit dose form of a composition is typically included in apharmaceutical composition that comprises a human unit dose of anacceptable carrier, in one embodiment an aqueous carrier, and isadministered in a volume/quantity that is known by those of skill in theart to be used for administration of such compositions to humans (see,e.g., Remington's Pharmaceutical Sciences, 17^(th) Edition, A. Gennaro,Editor, Mack Publishing Co., Easton, Pa., 1985). For example a peptidedose for initial immunization can be from about 1 to about 50,000 μg,generally 100-5,000 μg, for a 70 kg patient. For example, for nucleicacids an initial immunization may be performed using an expressionvector in the form of naked nucleic acid administered IM (or SC or ID)in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to1000 μg) can also be administered using a gene gun. Following anincubation period of 3-4 weeks, a booster dose is then administered. Thebooster can be recombinant fowlpox virus administered at a dose of 5-10⁷to 5×10⁹ pfu.

For antibodies, a treatment generally involves repeated administrationof the anti-24P4C12 antibody preparation, via an acceptable route ofadministration such as intravenous injection (IV), typically at a dosein the range of about 0.1 to about 10 mg/kg body weight. In general,doses in the range of 10-500 mg mAb per week are effective and welltolerated. Moreover, an initial loading dose of approximately 4 mg/kgpatient body weight IV, followed by weekly doses of about 2 mg/kg IV ofthe anti-24P4C12 mAb preparation represents an acceptable dosingregimen. As appreciated by those of skill in the art, various factorscan influence the ideal dose in a particular case. Such factors include,for example, half life of a composition, the binding affinity of an Ab,the immunogenicity of a substance, the degree of 24P4C12 expression inthe patient, the extent of circulating shed 24P4C12 antigen, the desiredsteady-state concentration level, frequency of treatment, and theinfluence of chemotherapeutic or other agents used in combination withthe treatment method of the invention, as well as the health status of aparticular patient. Non-limiting preferred human unit doses are, forexample, 500 μg-1 mg, 1 mg-50 mg, 50 mg-100 mg, 100 mg-200 mg, 200mg-300 mg, 400 mg-500 mg, 500 mg-600 mg, 600 mg-700 mg, 700 mg-800 mg,800 mg-900 mg, 900 mg-1g, or 1 mg-700 mg. In certain embodiments, thedose is in a range of 2-5 mg/kg body weight, e.g., with follow on weeklydoses of 1-3 mg/kg; 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/kg bodyweight followed, e.g., in two, three or four weeks by weekly doses;0.5-10 mg/kg body weight, e.g., followed in two, three or four weeks byweekly doses; 225, 250, 275, 300, 325, 350, 375, 400 mg m² of body areaweekly; 1-600 mg m² of body area weekly; 225-400 mg m² of body areaweekly; these does can be followed by weekly doses for 2, 3, 4, 5, 6, 7,8, 9, 19, 11, 12 or more weeks.

In one embodiment, human unit dose forms of polynucleotides comprise asuitable dosage range or effective amount that provides any therapeuticeffect. As appreciated by one of ordinary skill in the art a therapeuticeffect depends on a number of factors, including the sequence of thepolynucleotide, molecular weight of the polynucleotide and route ofadministration. Dosages are generally selected by the physician or otherhealth care professional in accordance with a variety of parametersknown in the art, such as severity of symptoms, history of the patientand the like. Generally, for a polynucleotide of about 20 bases, adosage range may be selected from, for example, an independentlyselected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kg up to anindependently selected upper limit, greater than the lower limit, ofabout 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, 3000,4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a dosemay be about any of the following: 0.1 to 100 mg/kg, 0.1 to 50 mg/kg,0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 mg/kg.Generally, parenteral routes of administration may require higher dosesof polynucleotide compared to more direct application to the nucleotideto diseased tissue, as do polynucleotides of increasing length.

In one embodiment, human unit dose forms of T-cells comprise a suitabledosage range or effective amount that provides any therapeutic effect.As appreciated by one of ordinary skill in the art, a therapeutic effectdepends on a number of factors. Dosages are generally selected by thephysician or other health care professional in accordance with a varietyof parameters known in the art, such as severity of symptoms, history ofthe patient and the like. A dose may be about 10⁴ cells to about 10⁶cells, about 10⁶ cells to about 10⁸ cells, about 10⁸ to about 10¹¹cells, or about 10⁸ to about 5×10¹⁰ cells. A dose may also about 10⁶cells/m² to about 10¹⁰ cells/m², or about 10⁶ cells/m² to about 10⁸cells/m²

Proteins(s) of the invention, and/or nucleic acids encoding theprotein(s), can also be administered via liposomes, which may also serveto: 1) target the proteins(s) to a particular tissue, such as lymphoidtissue; 2) to target selectively to diseases cells; or, 3) to increasethe half-life of the peptide composition. Liposomes include emulsions,foams, micelles, insoluble monolayers, liquid crystals, phospholipiddispersions, lamellar layers and the like. In these preparations, thepeptide to be delivered is incorporated as part of a liposome, alone orin conjunction with a molecule which binds to a receptor prevalent amonglymphoid cells, such as monoclonal antibodies which bind to the CD45antigen, or with other therapeutic or immunogenic compositions. Thus,liposomes either filled or decorated with a desired peptide of theinvention can be directed to the site of lymphoid cells, where theliposomes then deliver the peptide compositions. Liposomes for use inaccordance with the invention are formed from standard vesicle-forminglipids, which generally include neutral and negatively chargedphospholipids and a sterol, such as cholesterol. The selection of lipidsis generally guided by consideration of, e.g., liposome size, acidliability and stability of the liposomes in the blood stream. A varietyof methods are available for preparing liposomes, as described in, e.g.,Szoka, et al., Ann. Rev. Biophys. Bloeng. 9:467 (1980), and U.S. Pat.Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporatedinto the liposome can include, e.g., antibodies or fragments thereofspecific for cell surface determinants of the desired immune systemcells. A liposome suspension containing a peptide may be administeredintravenously, locally, topically, etc. in a dose which varies accordingto, inter alia, the manner of administration, the peptide beingdelivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more peptides of the invention, and morepreferably at a concentration of 25%-75%.

For aerosol administration, immunogenic peptides are preferably suppliedin finely divided form along with a surfactant and propellant. Typicalpercentages of peptides are about 0.01%-20% by weight, preferably about1%-10%. The surfactant must, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such agents are the estersor partial esters of fatty acids containing from about 6 to 22 carbonatoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Mixed esters, such as mixed or naturalglycerides may be employed. The surfactant may constitute about 0.1%-20%by weight of the composition, preferably about 0.25-5%. The balance ofthe composition is ordinarily propellant. A carrier can also beincluded, as desired, as with, e.g., lecithin for intranasal delivery.

XI.) DIAGNOSTIC AND PROGNOSTIC EMBODIMENTS OF 24P4C12

As disclosed herein, 24P4C12 polynucleotides, polypeptides, reactivecytotoxic T cells (CTL), reactive helper T cells (HTL) andanti-polypeptide antibodies are used in well known diagnostic,prognostic and therapeutic assays that examine conditions associatedwith dysregulated cell growth such as cancer, in particular the cancerslisted in Table I (see, e.g., both its specific pattern of tissueexpression as well as its overexpression in certain cancers as describedfor example in the Example entitled “Expression analysis of 24P4C12 innormal tissues, and patient specimens”).

24P4C12 can be analogized to a prostate associated antigen PSA, thearchetypal marker that has been used by medical practitioners for yearsto identify and monitor the presence of prostate cancer (see, e.g.,Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al., J.Urol. August; 162(2):293-306 (1999) and Fortier et al., J. Nat. CancerInst. 91(19): 1635-1640 (1999)). A variety of other diagnostic markersare also used in similar contexts including p53 and K-ras (see, e.g.,Tulchinsky et al., Int J Mol Med 1999 July 4(1):99-102 and Minimoto etal., Cancer Detect Prev 2000; 24(1):1-12). Therefore, this disclosure of24P4C12 polynucleotides and polypeptides (as well as 24P4C12polynucleotide probes and anti-24P4C12 antibodies used to identify thepresence of these molecules) and their properties allows skilledartisans to utilize these molecules in methods that are analogous tothose used, for example, in a variety of diagnostic assays directed toexamining conditions associated with cancer.

Typical embodiments of diagnostic methods which utilize the 24P4C12polynucleotides, polypeptides, reactive T cells and antibodies areanalogous to those methods from well-established diagnostic assays,which employ, e.g., PSA polynucleotides, polypeptides, reactive T cellsand antibodies. For example, just as PSA polynucleotides are used asprobes (for example in Northern analysis, see, e.g., Sharief et al.,Biochem. Mol. Biol. Int. 33(3):567-74 (1994)) and primers (for examplein PCR analysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190(2000)) to observe the presence and/or the level of PSA mRNAs in methodsof monitoring PSA overexpression or the metastasis of prostate cancers,the 24P4C12 polynucleotides described herein can be utilized in the sameway to detect 24P4C12 overexpression or the metastasis of prostate andother cancers expressing this gene. Alternatively, just as PSApolypeptides are used to generate antibodies specific for PSA which canthen be used to observe the presence and/or the level of PSA proteins inmethods to monitor PSA protein overexpression (see, e.g., Stephan etal., Urology 55(4):560-3 (2000)) or the metastasis of prostate cells(see, e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the24P4C12 polypeptides described herein can be utilized to generateantibodies for use in detecting 24P4C12 overexpression or the metastasisof prostate cells and cells of other cancers expressing this gene.

Specifically, because metastases involves the movement of cancer cellsfrom an organ of origin (such as the lung or prostate gland etc.) to adifferent area of the body (such as a lymph node), assays which examinea biological sample for the presence of cells expressing 24P4C12polynucleotides and/or polypeptides can be used to provide evidence ofmetastasis. For example, when a biological sample from tissue that doesnot normally contain 24P4C12-expressing cells (lymph node) is found tocontain 24P4C12-expressing cells such as the 24P4C12 expression seen inLAPC4 and LAPC9, xenografts isolated from lymph node and bonemetastasis, respectively, this finding is indicative of metastasis.

Alternatively 24P4C12 polynucleotides and/or polypeptides can be used toprovide evidence of cancer, for example, when cells in a biologicalsample that do not normally express 24P4C12 or express 24P4C12 at adifferent level are found to express 24P4C12 or have an increasedexpression of 24P4C12 (see, e.g., the 24P4C12 expression in the cancerslisted in Table I and in patient samples etc. shown in the accompanyingFigures). In such assays, artisans may further wish to generatesupplementary evidence of metastasis by testing the biological samplefor the presence of a second tissue restricted marker (in addition to24P4C12) such as PSA, PSCA etc. (see, e.g., Alanen et al., Pathol. Res.Pract. 192(3): 233-237 (1996)).

Just as PSA polynucleotide fragments and polynucleotide variants areemployed by skilled artisans for use in methods of monitoring PSA,24P4C12 polynucleotide fragments and polynucleotide variants are used inan analogous manner. In particular, typical PSA polynucleotides used inmethods of monitoring PSA are probes or primers which consist offragments of the PSA cDNA sequence. Illustrating this, primers used toPCR amplify a PSA polynucleotide must include less than the whole PSAsequence to function in the polymerase chain reaction. In the context ofsuch PCR reactions, skilled artisans generally create a variety ofdifferent polynucleotide fragments that can be used as primers in orderto amplify different portions of a polynucleotide of interest or tooptimize amplification reactions (see, e.g., Caetano-Anolles, G.Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., MethodsMol. Biol. 98:121-154 (1998)). An additional illustration of the use ofsuch fragments is provided in the Example entitled “Expression analysisof 24P4C12 in normal tissues, and patient specimens,” where a 24P4C12polynucleotide fragment is used as a probe to show the expression of24P4C12 RNAs in cancer cells. In addition, variant polynucleotidesequences are typically used as primers and probes for the correspondingmRNAs in PCR and Northern analyses (see, e.g., Sawai et al., FetalDiagn. Ther. 1996 November-December 11(6):407-13 and Current ProtocolsIn Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al.eds., 1995)). Polynucleotide fragments and variants are useful in thiscontext where they are capable of binding to a target polynucleotidesequence (e.g., a 24P4C12 polynucleotide shown in FIG. 2 or variantthereof) under conditions of high stringency.

Furthermore, PSA polypeptides which contain an epitope that can berecognized by an antibody or T cell that specifically binds to thatepitope are used in methods of monitoring PSA. 24P4C12 polypeptidefragments and polypeptide analogs or variants can also be used in ananalogous manner. This practice of using polypeptide fragments orpolypeptide variants to generate antibodies (such as anti-PSA antibodiesor T cells) is typical in the art with a wide variety of systems such asfusion proteins being used by practitioners (see, e.g., CurrentProtocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubelet al. eds., 1995). In this context, each epitope(s) functions toprovide the architecture with which an antibody or T cell is reactive.Typically, skilled artisans create a variety of different polypeptidefragments that can be used in order to generate immune responsesspecific for different portions of a polypeptide of interest (see, e.g.,U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it maybe preferable to utilize a polypeptide comprising one of the 24P4C12biological motifs discussed herein or a motif-bearing subsequence whichis readily identified by one of skill in the art based on motifsavailable in the art. Polypeptide fragments, variants or analogs aretypically useful in this context as long as they comprise an epitopecapable of generating an antibody or T cell specific for a targetpolypeptide sequence (e.g. a 24P4C12 polypeptide shown in FIG. 3).

As shown herein, the 24P4C12 polynucleotides and polypeptides (as wellas the 24P4C12 polynucleotide probes and anti-24P4C12 antibodies or Tcells used to identify the presence of these molecules) exhibit specificproperties that make them useful in diagnosing cancers such as thoselisted in Table I. Diagnostic assays that measure the presence of24P4C12 gene products, in order to evaluate the presence or onset of adisease condition described herein, such as prostate cancer, are used toidentify patients for preventive measures or further monitoring, as hasbeen done so successfully with PSA. Moreover, these materials satisfy aneed in the art for molecules having similar or complementarycharacteristics to PSA in situations where, for example, a definitediagnosis of metastasis of prostatic origin cannot be made on the basisof a test for PSA alone (see, e.g., Alanen et al., Pathol. Res. Pract.192(3): 233-237 (1996)), and consequently, materials such as 24P4C12polynucleotides and polypeptides (as well as the 24P4C12 polynucleotideprobes and anti-24P4C12 antibodies used to identify the presence ofthese molecules) need to be employed to confirm a metastases ofprostatic origin.

Finally, in addition to their use in diagnostic assays, the 24P4C12polynucleotides disclosed herein have a number of other utilities suchas their use in the identification of oncogenetic associated chromosomalabnormalities in the chromosomal region to which the 24P4C12 gene maps(see the Example entitled “Chromosomal Mapping of 24P4C12” below).Moreover, in addition to their use in diagnostic assays, the24P4C12-related proteins and polynucleotides disclosed herein have otherutilities such as their use in the forensic analysis of tissues ofunknown origin (see, e.g., Takahama K Forensic Sci Int 1996 Jun. 28;80(1-2): 63-9).

Additionally, 24P4C12-related proteins or polynucleotides of theinvention can be used to treat a pathologic condition characterized bythe over-expression of 24P4C12. For example, the amino acid or nucleicacid sequence of FIG. 2 or FIG. 3, or fragments of either, can be usedto generate an immune response to a 24P4C12 antigen. Antibodies or othermolecules that react with 24P4C12 can be used to modulate the functionof this molecule, and thereby provide a therapeutic benefit.

XII.) INHIBITION OF 24P4C12 PROTEIN FUNCTION

The invention includes various methods and compositions for inhibitingthe binding of 24P4C12 to its binding partner or its association withother protein(s) as well as methods for inhibiting 24P4C12 function.

XII.A.) Inhibition of 24P4C12 with Intracellular Antibodies

In one approach, a recombinant vector that encodes single chainantibodies that specifically bind to 24P4C12 are introduced into 24P4C12expressing cells via gene transfer technologies. Accordingly, theencoded single chain anti-24P4C12 antibody is expressed intracellularly,binds to 24P4C12 protein, and thereby inhibits its function. Methods forengineering such intracellular single chain antibodies are well known.Such intracellular antibodies, also known as “intrabodies”, arespecifically targeted to a particular compartment within the cell,providing control over where the inhibitory activity of the treatment isfocused. This technology has been successfully applied in the art (forreview, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodieshave been shown to virtually eliminate the expression of otherwiseabundant cell surface receptors (see, e.g., Richardson et al., 1995,Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol.Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337).

Single chain antibodies comprise the variable domains of the heavy andlight chain joined by a flexible linker polypeptide, and are expressedas a single polypeptide. Optionally, single chain antibodies areexpressed as a single chain variable region fragment joined to the lightchain constant region. Well-known intracellular trafficking signals areengineered into recombinant polynucleotide vectors encoding such singlechain antibodies in order to target precisely the intrabody to thedesired intracellular compartment. For example, intrabodies targeted tothe endoplasmic reticulum (ER) are engineered to incorporate a leaderpeptide and, optionally, a C-terminal ER retention signal, such as theKDEL amino acid motif. Intrabodies intended to exert activity in thenucleus are engineered to include a nuclear localization signal. Lipidmoieties are joined to intrabodies in order to tether the intrabody tothe cytosolic side of the plasma membrane. Intrabodies can also betargeted to exert function in the cytosol. For example, cytosolicintrabodies are used to sequester factors within the cytosol, therebypreventing them from being transported to their natural cellulardestination.

In one embodiment, intrabodies are used to capture 24P4C12 in thenucleus, thereby preventing its activity within the nucleus. Nucleartargeting signals are engineered into such 24P4C12 intrabodies in orderto achieve the desired targeting. Such 24P4C12 intrabodies are designedto bind specifically to a particular 24P4C12 domain. In anotherembodiment, cytosolic intrabodies that specifically bind to a 24P4C12protein are used to prevent 24P4C12 from gaining access to the nucleus,thereby preventing it from exerting any biological activity within thenucleus (e.g., preventing 24P4C12 from forming transcription complexeswith other factors).

In order to specifically direct the expression of such intrabodies toparticular cells, the transcription of the intrabody is placed under theregulatory control of an appropriate tumor-specific promoter and/orenhancer. In order to target intrabody expression specifically toprostate, for example, the PSA promoter and/or promoter/enhancer can beutilized (See, for example, U.S. Pat. No. 5,919,652 issued 6 Jul. 1999).

XII.B.) Inhibition of 24P4C12 with Recombinant Proteins

In another approach, recombinant molecules bind to 24P4C12 and therebyinhibit 24P4C12 function. For example, these recombinant moleculesprevent or inhibit 24P4C12 from accessing/binding to its bindingpartner(s) or associating with other protein(s). Such recombinantmolecules can, for example, contain the reactive part(s) of a 24P4C12specific antibody molecule. In a particular embodiment, the 24P4C12binding domain of a 24P4C12 binding partner is engineered into a dimericfusion protein, whereby the fusion protein comprises two 24P4C12 ligandbinding domains linked to the Fc portion of a human IgG, such as humanIgG1. Such IgG portion can contain, for example, the C_(H)2 and C_(H)3domains and the hinge region, but not the C_(H)1 domain. Such dimericfusion proteins are administered in soluble form to patients sufferingfrom a cancer associated with the expression of 24P4C12, whereby thedimeric fusion protein specifically binds to 24P4C12 and blocks 24P4C12interaction with a binding partner. Such dimeric fusion proteins arefurther combined into multimeric proteins using known antibody linkingtechnologies.

XII.C.) Inhibition of 24P4C12 Transcription or Translation

The present invention also comprises various methods and compositionsfor inhibiting the transcription of the 24P4C12 gene. Similarly, theinvention also provides methods and compositions for inhibiting thetranslation of 24P4C12 mRNA into protein.

In one approach, a method of inhibiting the transcription of the 24P4C12gene comprises contacting the 24P4C12 gene with a 24P4C12 antisensepolynucleotide. In another approach, a method of inhibiting 24P4C12 mRNAtranslation comprises contacting a 24P4C12 mRNA with an antisensepolynucleotide. In another approach, a 24P4C12 specific ribozyme is usedto cleave a 24P4C12 message, thereby inhibiting translation. Suchantisense and ribozyme based methods can also be directed to theregulatory regions of the 24P4C12 gene, such as 24P4C12 promoter and/orenhancer elements. Similarly, proteins capable of inhibiting a 24P4C12gene transcription factor are used to inhibit 24P4C12 mRNAtranscription. The various polynucleotides and compositions useful inthe aforementioned methods have been described above. The use ofantisense and ribozyme molecules to inhibit transcription andtranslation is well known in the art.

Other factors that inhibit the transcription of 24P4C12 by interferingwith 24P4C12 transcriptional activation are also useful to treat cancersexpressing 24P4C12. Similarly, factors that interfere with 24P4C12processing are useful to treat cancers that express 24P4C12. Cancertreatment methods utilizing such factors are also within the scope ofthe invention.

XII.D.) General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies can be used to delivertherapeutic polynucleotide molecules to tumor cells synthesizing 24P4C12(i.e., antisense, ribozyme, polynucleotides encoding intrabodies andother 24P4C12 inhibitory molecules). A number of gene therapy approachesare known in the art. Recombinant vectors encoding 24P4C12 antisensepolynucleotides, ribozymes, factors capable of interfering with 24P4C12transcription, and so forth, can be delivered to target tumor cellsusing such gene therapy approaches.

The above therapeutic approaches can be combined with any one of a widevariety of surgical, chemotherapy or radiation therapy regimens. Thetherapeutic approaches of the invention can enable the use of reduceddosages of chemotherapy (or other therapies) and/or less frequentadministration, an advantage for all patients and particularly for thosethat do not tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense,ribozyme, intrabody), or a combination of such compositions, can beevaluated using various in vitro and in vivo assay systems. In vitroassays that evaluate therapeutic activity include cell growth assays,soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of 24P4C12 to a bindingpartner, etc.

In vivo, the effect of a 24P4C12 therapeutic composition can beevaluated in a suitable animal model. For example, xenogenic prostatecancer models can be used, wherein human prostate cancer explants orpassaged xenograft tissues are introduced into immune compromisedanimals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine3: 402-408). For example, PCT Patent Application WO98/16628 and U.S.Pat. No. 6,107,540 describe various xenograft models of human prostatecancer capable of recapitulating the development of primary tumors,micrometastasis, and the formation of osteoblastic metastasescharacteristic of late stage disease. Efficacy can be predicted usingassays that measure inhibition of tumor formation, tumor regression ormetastasis, and the like.

In vivo assays that evaluate the promotion of apoptosis are useful inevaluating therapeutic compositions. In one embodiment, xenografts fromtumor bearing mice treated with the therapeutic composition can beexamined for the presence of apoptotic foci and compared to untreatedcontrol xenograft-bearing mice. The extent to which apoptotic foci arefound in the tumors of the treated mice provides an indication of thetherapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16^(th) Edition, A. Osal., Ed.,1980).

Therapeutic formulations can be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations can be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water (containing for example, benzyl alcoholpreservative) or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancer,and will generally depend on a number of other factors appreciated inthe art.

XIII.) IDENTIFICATION, CHARACTERIZATION AND USE OF MODULATORS OF 24P4C12

Methods to Identify and Use Modulators

In one embodiment, screening is performed to identify modulators thatinduce or suppress a particular expression profile, suppress or inducespecific pathways, preferably generating the associated phenotypethereby. In another embodiment, having identified differentiallyexpressed genes important in a particular state; screens are performedto identify modulators that alter expression of individual genes, eitherincrease or decrease. In another embodiment, screening is performed toidentify modulators that alter a biological function of the expressionproduct of a differentially expressed gene. Again, having identified theimportance of a gene in a particular state, screens are performed toidentify agents that bind and/or modulate the biological activity of thegene product.

In addition, screens are done for genes that are induced in response toa candidate agent. After identifying a modulator (one that suppresses acancer expression pattern leading to a normal expression pattern, or amodulator of a cancer gene that leads to expression of the gene as innormal tissue) a screen is performed to identify genes that arespecifically modulated in response to the agent. Comparing expressionprofiles between normal tissue and agent-treated cancer tissue revealsgenes that are not expressed in normal tissue or cancer tissue, but areexpressed in agent treated tissue, and vice versa. These agent-specificsequences are identified and used by methods described herein for cancergenes or proteins. In particular these sequences and the proteins theyencode are used in marking or identifying agent-treated cells. Inaddition, antibodies are raised against the agent-induced proteins andused to target novel therapeutics to the treated cancer tissue sample.

Modulator-Related Identification and Screening Assays:

Gene Expression-Related Assays

Proteins, nucleic acids, and antibodies of the invention are used inscreening assays. The cancer-associated proteins, antibodies, nucleicacids, modified proteins and cells containing these sequences are usedin screening assays, such as evaluating the effect of drug candidates ona “gene expression profile,” expression profile of polypeptides oralteration of biological function. In one embodiment, the expressionprofiles are used, preferably in conjunction with high throughputscreening techniques to allow monitoring for expression profile genesafter treatment with a candidate agent (e.g., Davis, G F, et al, J BiolScreen 7:69 (2002); Zlokarnik, et al., Science 279:84-8 (1998); Heid,Genome Res 6:986-94, 1996).

The cancer proteins, antibodies, nucleic acids, modified proteins andcells containing the native or modified cancer proteins or genes areused in screening assays. That is, the present invention comprisesmethods for screening for compositions which modulate the cancerphenotype or a physiological function of a cancer protein of theinvention. This is done on a gene itself or by evaluating the effect ofdrug candidates on a “gene expression profile” or biological function.In one embodiment, expression profiles are used, preferably inconjunction with high throughput screening techniques to allowmonitoring after treatment with a candidate agent, see Zlokamik, supra.

A variety of assays are executed directed to the genes and proteins ofthe invention. Assays are run on an individual nucleic acid or proteinlevel. That is, having identified a particular gene as up regulated incancer, test compounds are screened for the ability to modulate geneexpression or for binding to the cancer protein of the invention.“Modulation” in this context includes an increase or a decrease in geneexpression. The preferred amount of modulation will depend on theoriginal change of the gene expression in normal versus tissueundergoing cancer, with changes of at least 10%, preferably 50%, morepreferably 100-300%, and in some embodiments 300-1000% or greater. Thus,if a gene exhibits a 4-fold increase in cancer tissue compared to normaltissue, a decrease of about four-fold is often desired; similarly, a10-fold decrease in cancer tissue compared to normal tissue a targetvalue of a 10-fold increase in expression by the test compound is oftendesired. Modulators that exacerbate the type of gene expression seen incancer are also useful, e.g., as an upregulated target in furtheranalyses.

The amount of gene expression is monitored using nucleic acid probes andthe quantification of gene expression levels, or, alternatively, a geneproduct itself is monitored, e.g., through the use of antibodies to thecancer protein and standard immunoassays. Proteomics and separationtechniques also allow for quantification of expression.

Expression Monitoring to Identify Compounds that Modify Gene Expression

In one embodiment, gene expression monitoring, i.e., an expressionprofile, is monitored simultaneously for a number of entities. Suchprofiles will typically involve one or more of the genes of FIG. 2. Inthis embodiment, e.g., cancer nucleic acid probes are attached tobiochips to detect and quantify cancer sequences in a particular cell.Alternatively, PCR can be used. Thus, a series, e.g., wells of amicrotiter plate, can be used with dispensed primers in desired wells. APCR reaction can then be performed and analyzed for each well.

Expression monitoring is performed to identify compounds that modify theexpression of one or more cancer-associated sequences, e.g., apolynucleotide sequence set out in FIG. 2. Generally, a test modulatoris added to the cells prior to analysis. Moreover, screens are alsoprovided to identify agents that modulate cancer, modulate cancerproteins of the invention, bind to a cancer protein of the invention, orinterfere with the binding of a cancer protein of the invention and anantibody or other binding partner.

In one embodiment, high throughput screening methods involve providing alibrary containing a large number of potential therapeutic compounds(candidate compounds). Such “combinatorial chemical libraries” are thenscreened in one or more assays to identify those library members(particular chemical species or subclasses) that display a desiredcharacteristic activity. The compounds thus identified can serve asconventional “lead compounds,” as compounds for screening, or astherapeutics.

In certain embodiments, combinatorial libraries of potential modulatorsare screened for an ability to bind to a cancer polypeptide or tomodulate activity. Conventionally, new chemical entities with usefulproperties are generated by identifying a chemical compound (called a“lead compound”) with some desirable property or activity, e.g.,inhibiting activity, creating variants of the lead compound, andevaluating the property and activity of those variant compounds. Often,high throughput screening (HTS) methods are employed for such ananalysis.

As noted above, gene expression monitoring is conveniently used to testcandidate modulators (e.g., protein, nucleic acid or small molecule).After the candidate agent has been added and the cells allowed toincubate for a period, the sample containing a target sequence to beanalyzed is, e.g., added to a biochip.

If required, the target sequence is prepared using known techniques. Forexample, a sample is treated to lyse the cells, using known lysisbuffers, electroporation, etc., with purification and/or amplificationsuch as PCR performed as appropriate. For example, an in vitrotranscription with labels covalently attached to the nucleotides isperformed. Generally, the nucleic acids are labeled with biotin-FITC orPE, or with cy3 or cy5.

The target sequence can be labeled with, e.g., a fluorescent, achemiluminescent, a chemical, or a radioactive signal, to provide ameans of detecting the target sequence's specific binding to a probe.The label also can be an enzyme, such as alkaline phosphatase orhorseradish peroxidase, which when provided with an appropriatesubstrate produces a product that is detected. Alternatively, the labelis a labeled compound or small molecule, such as an enzyme inhibitor,that binds but is not catalyzed or altered by the enzyme. The label alsocan be a moiety or compound, such as, an epitope tag or biotin whichspecifically binds to streptavidin. For the example of biotin, thestreptavidin is labeled as described above, thereby, providing adetectable signal for the bound target sequence. Unbound labeledstreptavidin is typically removed prior to analysis.

As will be appreciated by those in the art, these assays can be directhybridization assays or can comprise “sandwich assays”, which includethe use of multiple probes, as is generally outlined in U.S. Pat. Nos.5,681,702; 5,597,909; 5,545,730; 5,594,117; 5,591,584; 5,571,670;5,580,731; 5,571,670; 5,591,584; 5,624,802; 5,635,352; 5,594,118;5,359,100; 5,124,246; and 5,681,697. In this embodiment, in general, thetarget nucleic acid is prepared as outlined above, and then added to thebiochip comprising a plurality of nucleic acid probes, under conditionsthat allow the formation of a hybridization complex.

A variety of hybridization conditions are used in the present invention,including high, moderate and low stringency conditions as outlinedabove. The assays are generally run under stringency conditions whichallow formation of the label probe hybridization complex only in thepresence of target. Stringency can be controlled by altering a stepparameter that is a thermodynamic variable, including, but not limitedto, temperature, formamide concentration, salt concentration, chaotropicsalt concentration pH, organic solvent concentration, etc. Theseparameters may also be used to control non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697. Thus, it can be desirableto perform certain steps at higher stringency conditions to reducenon-specific binding.

The reactions outlined herein can be accomplished in a variety of ways.Components of the reaction can be added simultaneously, or sequentially,in different orders, with preferred embodiments outlined below. Inaddition, the reaction may include a variety of other reagents. Theseinclude salts, buffers, neutral proteins, e.g. albumin, detergents, etc.which can be used to facilitate optimal hybridization and detection,and/or reduce nonspecific or background interactions. Reagents thatotherwise improve the efficiency of the assay, such as proteaseinhibitors, nuclease inhibitors, anti-microbial agents, etc., may alsobe used as appropriate, depending on the sample preparation methods andpurity of the target. The assay data are analyzed to determine theexpression levels of individual genes, and changes in expression levelsas between states, forming a gene expression profile.

Biological Activity-Related Assays

The invention provides methods identify or screen for a compound thatmodulates the activity of a cancer-related gene or protein of theinvention. The methods comprise adding a test compound, as definedabove, to a cell comprising a cancer protein of the invention. The cellscontain a recombinant nucleic acid that encodes a cancer protein of theinvention. In another embodiment, a library of candidate agents istested on a plurality of cells.

In one aspect, the assays are evaluated in the presence or absence orprevious or subsequent exposure of physiological signals, e.g. hormones,antibodies, peptides, antigens, cytokines, growth factors, actionpotentials, pharmacological agents including chemotherapeutics,radiation, carcinogenics, or other cells (i.e., cell-cell contacts). Inanother example, the determinations are made at different stages of thecell cycle process. In this way, compounds that modulate genes orproteins of the invention are identified. Compounds with pharmacologicalactivity are able to enhance or interfere with the activity of thecancer protein of the invention. Once identified, similar structures areevaluated to identify critical structural features of the compound.

In one embodiment, a method of modulating (e.g., inhibiting) cancer celldivision is provided; the method comprises administration of a cancermodulator. In another embodiment, a method of modulating (e.g.,inhibiting) cancer is provided; the method comprises administration of acancer modulator. In a further embodiment, methods of treating cells orindividuals with cancer are provided; the method comprisesadministration of a cancer modulator.

In one embodiment, a method for modulating the status of a cell thatexpresses a gene of the invention is provided. As used herein statuscomprises such art-accepted parameters such as growth, proliferation,survival, function, apoptosis, senescence, location, enzymatic activity,signal transduction, etc. of a cell. In one embodiment, a cancerinhibitor is an antibody as discussed above. In another embodiment, thecancer inhibitor is an antisense molecule. A variety of cell growth,proliferation, and metastasis assays are known to those of skill in theart, as described herein.

High Throughput Screening to Identify Modulators

The assays to identify suitable modulators are amenable to highthroughput screening. Preferred assays thus detect enhancement orinhibition of cancer gene transcription, inhibition or enhancement ofpolypeptide expression, and inhibition or enhancement of polypeptideactivity.

In one embodiment, modulators evaluated in high throughput screeningmethods are proteins, often naturally occurring proteins or fragments ofnaturally occurring proteins. Thus, e.g., cellular extracts containingproteins, or random or directed digests of proteinaceous cellularextracts, are used. In this way, libraries of proteins are made forscreening in the methods of the invention. Particularly preferred inthis embodiment are libraries of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred. Particularly useful test compound will be directedto the class of proteins to which the target belongs, e.g., substratesfor enzymes, or ligands and receptors.

Use of Soft Agar Growth and Colony Formation to Identify andCharacterize Modulators

Normal cells require a solid substrate to attach and grow. When cellsare transformed, they lose this phenotype and grow detached from thesubstrate. For example, transformed cells can grow in stirred suspensionculture or suspended in semi-solid media, such as semi-solid or softagar. The transformed cells, when transfected with tumor suppressorgenes, can regenerate normal phenotype and once again require a solidsubstrate to attach to and grow. Soft agar growth or colony formation inassays are used to identify modulators of cancer sequences, which whenexpressed in host cells, inhibit abnormal cellular proliferation andtransformation. A modulator reduces or eliminates the host cells'ability to grow suspended in solid or semisolid media, such as agar.

Techniques for soft agar growth or colony formation in suspension assaysare described in Freshney, Culture of Animal Cells a Manual of BasicTechnique (3rd ed., 1994). See also, the methods section of Garkavtsevet al. (1996), supra.

Evaluation of Contact Inhibition and Growth Density Limitation toIdentify and Characterize Modulators

Normal cells typically grow in a flat and organized pattern in cellculture until they touch other cells. When the cells touch one another,they are contact inhibited and stop growing. Transformed cells, however,are not contact inhibited and continue to grow to high densities indisorganized foci. Thus, transformed cells grow to a higher saturationdensity than corresponding normal cells. This is detectedmorphologically by the formation of a disoriented monolayer of cells orcells in foci. Alternatively, labeling index with (³H)-thymidine atsaturation density is used to measure density limitation of growth,similarly an MTT or Alamar blue assay will reveal proliferation capacityof cells and the ability of modulators to affect same. See Freshney(1994), supra. Transformed cells, when transfected with tumor suppressorgenes, can regenerate a normal phenotype and become contact inhibitedand would grow to a lower density.

In this assay, labeling index with 3H)-thymidine at saturation densityis a preferred method of measuring density limitation of growth.Transformed host cells are transfected with a cancer-associated sequenceand are grown for 24 hours at saturation density in non-limiting mediumconditions. The percentage of cells labeling with (³H)-thymidine isdetermined by incorporated cpm.

Contact independent growth is used to identify modulators of cancersequences, which had led to abnormal cellular proliferation andtransformation. A modulator reduces or eliminates contact independentgrowth, and returns the cells to a normal phenotype.

Evaluation of Growth Factor or Serum Dependence to Identity andCharacterize Modulators

Transformed cells have lower serum dependence than their normalcounterparts (see, e.g., Temin, J. Natl. Cancer Inst. 37:167-175 (1966);Eagle et al., J. Exp. Med. 131:836-879 (1970)); Freshney, supra. This isin part due to release of various growth factors by the transformedcells. The degree of growth factor or serum dependence of transformedhost cells can be compared with that of control. For example, growthfactor or serum dependence of a cell is monitored in methods to identifyand characterize compounds that modulate cancer-associated sequences ofthe invention.

Use of Tumor-Specific Marker Levels to Identify and CharacterizeModulators

Tumor cells release an increased amount of certain factors (hereinafter“tumor specific markers”) than their normal counterparts. For example,plasminogen activator (PA) is released from human glioma at a higherlevel than from normal brain cells (see, e.g., Gullino, Angiogenesis,Tumor Vascularization, and Potential Interference with Tumor Growth, inBiological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985)).Similarly, Tumor Angiogenesis Factor (TAF) is released at a higher levelin tumor cells than their normal counterparts. See, e.g., Folkman,Angiogenesis and Cancer, Sem Cancer Biol. (1992)), while bFGF isreleased from endothelial tumors (Ensoli, B et al).

Various techniques which measure the release of these factors aredescribed in Freshney (1994), supra. Also, see, Unkless et al., J. Biol.Chem. 249:4295-4305 (1974); Strickland & Beers, J. Biol. Chem.251:5694-5702 (1976); Whur et al., Br. J. Cancer 42:305 312 (1980);Gullino, Angiogenesis, Tumor Vascularization, and Potential Interferencewith Tumor Growth, in Biological Responses in Cancer, pp. 178-184(Mihich (ed.) 1985); Freshney, Anticancer Res. 5:111-130 (1985). Forexample, tumor specific marker levels are monitored in methods toidentify and characterize compounds that modulate cancer-associatedsequences of the invention.

Invasiveness into Matrigel to Identify and Characterize Modulators

The degree of invasiveness into Matrigel or an extracellular matrixconstituent can be used as an assay to identify and characterizecompounds that modulate cancer associated sequences. Tumor cells exhibita positive correlation between malignancy and invasiveness of cells intoMatrigel or some other extracellular matrix constituent. In this assay,tumorigenic cells are typically used as host cells. Expression of atumor suppressor gene in these host cells would decrease invasiveness ofthe host cells. Techniques described in Cancer Res. 1999; 59:6010;Freshney (1994), supra, can be used. Briefly, the level of invasion ofhost cells is measured by using filters coated with Matrigel or someother extracellular matrix constituent. Penetration into the gel, orthrough to the distal side of the filter, is rated as invasiveness, andrated histologically by number of cells and distance moved, or byprelabeling the cells with ¹²⁵I and counting the radioactivity on thedistal side of the filter or bottom of the dish. See, e.g., Freshney(1984), supra.

Evaluation of Tumor Growth In Vivo to Identify and CharacterizeModulators

Effects of cancer-associated sequences on cell growth are tested intransgenic or immune-suppressed organisms. Transgenic organisms areprepared in a variety of art-accepted ways. For example, knock-outtransgenic organisms, e.g., mammals such as mice, are made, in which acancer gene is disrupted or in which a cancer gene is inserted.Knock-out transgenic mice are made by insertion of a marker gene orother heterologous gene into the endogenous cancer gene site in themouse genome via homologous recombination. Such mice can also be made bysubstituting the endogenous cancer gene with a mutated version of thecancer gene, or by mutating the endogenous cancer gene, e.g., byexposure to carcinogens.

To prepare transgenic chimeric animals, e.g., mice, a DNA construct isintroduced into the nuclei of embryonic stem cells. Cells containing thenewly engineered genetic lesion are injected into a host mouse embryo,which is re-implanted into a recipient female. Some of these embryosdevelop into chimeric mice that possess germ cells some of which arederived from the mutant cell line. Therefore, by breeding the chimericmice it is possible to obtain a new line of mice containing theintroduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288(1989)). Chimeric mice can be derived according to U.S. Pat. No.6,365,797, issued 2 Apr. 2002; U.S. Pat. No. 6,107,540 issued 22 Aug.2000; Hogan et al., Manipulating the Mouse Embryo: A laboratory Manual,Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and EmbryonicStem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington,D.C., (1987).

Alternatively, various immune-suppressed or immune-deficient hostanimals can be used. For example, a genetically athymic “nude” mouse(see, e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921 (1974)), aSCID mouse, a thymectornized mouse, or an irradiated mouse (see, e.g.,Bradley et al., Br. J. Cancer 38:263 (1978); Selby et al., Br. J. Cancer41:52 (1980)) can be used as a host. Transplantable tumor cells(typically about 10⁶ cells) injected into isogenic hosts produceinvasive tumors in a high proportion of cases, while normal cells ofsimilar origin will not. In hosts which developed invasive tumors, cellsexpressing cancer-associated sequences are injected subcutaneously ororthotopically. Mice are then separated into groups, including controlgroups and treated experimental groups) e.g. treated with a modulator).After a suitable length of time, preferably 4-8 weeks, tumor growth ismeasured (e.g., by volume or by its two largest dimensions, or weight)and compared to the control. Tumors that have statistically significantreduction (using, e.g., Student's T test) are said to have inhibitedgrowth.

In Vitro Assays to Identify and Characterize Modulators

Assays to identify compounds with modulating activity can be performedin vitro. For example, a cancer polypeptide is first contacted with apotential modulator and incubated for a suitable amount of time, e.g.,from 0.5 to 48 hours. In one embodiment, the cancer polypeptide levelsare determined in vitro by measuring the level of protein or mRNA. Thelevel of protein is measured using immunoassays such as Westernblotting, ELISA and the like with an antibody that selectively binds tothe cancer polypeptide or a fragment thereof. For measurement of mRNA,amplification, e.g., using PCR, LCR, or hybridization assays, e.g.,Northern hybridization, RNAse protection, dot blotting, are preferred.The level of protein or mRNA is detected using directly or indirectlylabeled detection agents, e.g., fluorescently or radioactively labelednucleic acids, radioactively or enzymatically labeled antibodies, andthe like, as described herein.

Alternatively, a reporter gene system can be devised using a cancerprotein promoter operably linked to a reporter gene such as luciferase,green fluorescent protein, CAT, or P-gal. The reporter construct istypically transfected into a cell. After treatment with a potentialmodulator, the amount of reporter gene transcription, translation, oractivity is measured according to standard techniques known to those ofskill in the art (Davis G F, supra; Gonzalez, J. & Negulescu, P. Curr.Opin. Biotechnol. 1998: 9:624).

As outlined above, in vitro screens are done on individual genes andgene products. That is, having identified a particular differentiallyexpressed gene as important in a particular state, screening ofmodulators of the expression of the gene or the gene product itself isperformed.

In one embodiment, screening for modulators of expression of specificgene(s) is performed. Typically, the expression of only one or a fewgenes is evaluated. In another embodiment, screens are designed to firstfind compounds that bind to differentially expressed proteins. Thesecompounds are then evaluated for the ability to modulate differentiallyexpressed activity. Moreover, once initial candidate compounds areidentified, variants can be further screened to better evaluatestructure activity relationships.

Binding Assays to Identify and Characterize Modulators

In binding assays in accordance with the invention, a purified orisolated gene product of the invention is generally used. For example,antibodies are generated to a protein of the invention, and immunoassaysare run to determine the amount and/or location of protein.Alternatively, cells comprising the cancer proteins are used in theassays.

Thus, the methods comprise combining a cancer protein of the inventionand a candidate compound such as a ligand, and determining the bindingof the compound to the cancer protein of the invention. Preferredembodiments utilize the human cancer protein; animal models of humandisease of can also be developed and used. Also, other analogousmammalian proteins also can be used as appreciated by those of skill inthe art. Moreover, in some embodiments variant or derivative cancerproteins are used.

Generally, the cancer protein of the invention, or the ligand, isnon-diffusibly bound to an insoluble support. The support can, e.g., beone having isolated sample receiving areas (a microtiter plate, anarray, etc.). The insoluble supports can be made of any composition towhich the compositions can be bound, is readily separated from solublematerial, and is otherwise compatible with the overall method ofscreening. The surface of such supports can be solid or porous and ofany convenient shape.

Examples of suitable insoluble supports include microtiter plates,arrays, membranes and beads. These are typically made of glass, plastic(e.g., polystyrene), polysaccharide, nylon, nitrocellulose, or Teflon™,etc. Microtiter plates and arrays are especially convenient because alarge number of assays can be carried out simultaneously, using smallamounts of reagents and samples. The particular manner of binding of thecomposition to the support is not crucial so long as it is compatiblewith the reagents and overall methods of the invention, maintains theactivity of the composition and is nondiffusable. Preferred methods ofbinding include the use of antibodies which do not sterically blockeither the ligand binding site or activation sequence when attaching theprotein to the support, direct binding to “sticky” or ionic supports,chemical crosslinking, the synthesis of the protein or agent on thesurface, etc. Following binding of the protein or ligand/binding agentto the support, excess unbound material is removed by washing. Thesample receiving areas may then be blocked through incubation withbovine serum albumin (BSA), casein or other innocuous protein or othermoiety.

Once a cancer protein of the invention is bound to the support, and atest compound is added to the assay. Alternatively, the candidatebinding agent is bound to the support and the cancer protein of theinvention is then added. Binding agents include specific antibodies,non-natural binding agents identified in screens of chemical libraries,peptide analogs, etc.

Of particular interest are assays to identify agents that have a lowtoxicity for human cells. A wide variety of assays can be used for thispurpose, including proliferation assays, cAMP assays, labeled in vitroprotein-protein binding assays, electrophoretic mobility shift assays,immunoassays for protein binding, functional assays (phosphorylationassays, etc.) and the like.

A determination of binding of the test compound (ligand, binding agent,modulator, etc.) to a cancer protein of the invention can be done in anumber of ways. The test compound can be labeled, and binding determineddirectly, e.g., by attaching all or a portion of the cancer protein ofthe invention to a solid support, adding a labeled candidate compound(e.g., a fluorescent label), washing off excess reagent, and determiningwhether the label is present on the solid support. Various blocking andwashing steps can be utilized as appropriate.

In certain embodiments, only one of the components is labeled, e.g., aprotein of the invention or ligands labeled. Alternatively, more thanone component is labeled with different labels, e.g., I¹²⁵, for theproteins and a fluorophor for the compound. Proximity reagents, e.g.,quenching or energy transfer reagents are also useful.

Competitive Binding to Identify and Characterize Modulators

In one embodiment, the binding of the “test compound” is determined bycompetitive binding assay with a “competitor.” The competitor is abinding moiety that binds to the target molecule (e.g., a cancer proteinof the invention). Competitors include compounds such as antibodies,peptides, binding partners, ligands, etc. Under certain circumstances,the competitive binding between the test compound and the competitordisplaces the test compound. In one embodiment, the test compound islabeled. Either the test compound, the competitor, or both, is added tothe protein for a time sufficient to allow binding. Incubations areperformed at a temperature that facilitates optimal activity, typicallybetween four and 40° C. Incubation periods are typically optimized,e.g., to facilitate rapid high throughput screening; typically betweenzero and one hour will be sufficient. Excess reagent is generallyremoved or washed away. The second component is then added, and thepresence or absence of the labeled component is followed, to indicatebinding.

In one embodiment, the competitor is added first, followed by the testcompound. Displacement of the competitor is an indication that the testcompound is binding to the cancer protein and thus is capable of bindingto, and potentially modulating, the activity of the cancer protein. Inthis embodiment, either component can be labeled. Thus, e.g., if thecompetitor is labeled, the presence of label in the post-test compoundwash solution indicates displacement by the test compound.Alternatively, if the test compound is labeled, the presence of thelabel on the support indicates displacement.

In an alternative embodiment, the test compound is added first, withincubation and washing, followed by the competitor. The absence ofbinding by the competitor indicates that the test compound binds to thecancer protein with higher affinity than the competitor. Thus, if thetest compound is labeled, the presence of the label on the support,coupled with a lack of competitor binding, indicates that the testcompound binds to and thus potentially modulates the cancer protein ofthe invention.

Accordingly, the competitive binding methods comprise differentialscreening to identity agents that are capable of modulating the activityof the cancer proteins of the invention. In this embodiment, the methodscomprise combining a cancer protein and a competitor in a first sample.A second sample comprises a test compound, the cancer protein, and acompetitor. The binding of the competitor is determined for bothsamples, and a change, or difference in binding between the two samplesindicates the presence of an agent capable of binding to the cancerprotein and potentially modulating its activity. That is, if the bindingof the competitor is different in the second sample relative to thefirst sample, the agent is capable of binding to the cancer protein.

Alternatively, differential screening is used to identify drugcandidates that bind to the native cancer protein, but cannot bind tomodified cancer proteins. For example the structure of the cancerprotein is modeled and used in rational drug design to synthesize agentsthat interact with that site, agents which generally do not bind tosite-modified proteins. Moreover, such drug candidates that affect theactivity of a native cancer protein are also identified by screeningdrugs for the ability to either enhance or reduce the activity of suchproteins.

Positive controls and negative controls can be used in the assays.Preferably control and test samples are performed in at least triplicateto obtain statistically significant results. Incubation of all samplesoccurs for a time sufficient to allow for the binding of the agent tothe protein. Following incubation, samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined. For example, where a radiolabel is employed,the samples can be counted in a scintillation counter to determine theamount of bound compound.

A variety of other reagents can be included in the screening assays.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc. which are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,can be used. The mixture of components is added in an order thatprovides for the requisite binding.

Use of Polynucleotides to Down-regulate or Inhibit a Protein of theInvention.

Polynucleotide modulators of cancer can be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand-binding molecule, as described in WO 91/04753. Suitableligand-binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell. Alternatively, a polynucleotide modulator ofcancer can be introduced into a cell containing the target nucleic acidsequence, e.g., by formation of a polynucleotide-lipid complex, asdescribed in WO 90/10448. It is understood that the use of antisensemolecules or knock out and knock in models may also be used in screeningassays as discussed above, in addition to methods of treatment.

Inhibitory and Antisense Nucleotides

In certain embodiments, the activity of a cancer-associated protein isdown-regulated, or entirely inhibited, by the use of antisensepolynucleotide or inhibitory small nuclear RNA (snRNA), i.e., a nucleicacid complementary to, and which can preferably hybridize specificallyto, a coding mRNA nucleic acid sequence, e.g., a cancer protein of theinvention, mRNA, or a subsequence thereof. Binding of the antisensepolynucleotide to the mRNA reduces the translation and/or stability ofthe mRNA.

In the context of this invention, antisense polynucleotides can comprisenaturally occurring nucleotides, or synthetic species formed fromnaturally occurring subunits or their close homologs. Antisensepolynucleotides may also have altered sugar moieties or inter-sugarlinkages. Exemplary among these are the phosphorothioate and othersulfur containing species which are known for use in the art. Analogsare comprised by this invention so long as they function effectively tohybridize with nucleotides of the invention. See, e.g., IsisPharmaceuticals, Carlsbad, Calif.; Sequitor, Inc., Natick, Mass.

Such antisense polynucleotides can readily be synthesized usingrecombinant means, or can be synthesized in vitro. Equipment for suchsynthesis is sold by several vendors, including Applied Biosystems. Thepreparation of other oligonucleotides such as phosphorothioates andalkylated derivatives is also well known to those of skill in the art.

Antisense molecules as used herein include antisense or senseoligonucleotides. Sense oligonucleotides can, e.g., be employed to blocktranscription by binding to the anti-sense strand. The antisense andsense oligonucleotide comprise a single stranded nucleic acid sequence(either RNA or DNA) capable of binding to target mRNA (sense) or DNA(antisense) sequences for cancer molecules. Antisense or senseoligonucleotides, according to the present invention, comprise afragment generally at least about 12 nucleotides, preferably from about12 to 30 nucleotides. The ability to derive an antisense or a senseoligonucleotide, based upon a cDNA sequence encoding a given protein isdescribed in, e.g., Stein & Cohen (Cancer Res. 48:2659 (1988 and van derKrol et al. (BioTechniques 6:958 (1988)).

Ribozymes

In addition to antisense polynucleotides, ribozymes can be used totarget and inhibit transcription of cancer-associated nucleotidesequences. A ribozyme is an RNA molecule that catalytically cleavesother RNA molecules. Different kinds of ribozymes have been described,including group I ribozymes, hammerhead ribozymes, hairpin ribozymes,RNase P, and axhead ribozymes (see, e.g., Castanotto et al., Adv. inPharmacology 25: 289-317 (1994) for a general review of the propertiesof different ribozymes).

The general features of hairpin ribozymes are described, e.g., in Hampelet al., Nucl. Acids Res. 18:299-304 (1990); European Patent PublicationNo. 0360257; U.S. Pat. No. 5,254,678. Methods of preparing are wellknown to those of skill in the art (see, e.g., WO 94/26877; Ojwang etal., Proc. Natl. Acad. Sci. USA 90:6340-6344 (1993); Yamada et al.,Human Gene Therapy 1:39-45 (1994); Leavitt et al., Proc. Natl. Acad.Sci. USA 92:699-703 (1995); Leavitt et al., Human Gene Therapy 5:1151-120 (1994); and Yamada et al., Virology 205: 121-126 (1994)).

Use of Modulators in Phenotypic Screening

In one embodiment, a test compound is administered to a population ofcancer cells, which have an associated cancer expression profile. By“administration” or “contacting” herein is meant that the modulator isadded to the cells in such a manner as to allow the modulator to actupon the cell, whether by uptake and intracellular action, or by actionat the cell surface. In some embodiments, a nucleic acid encoding aproteinaceous agent (i.e., a peptide) is put into a viral construct suchas an adenoviral or retroviral construct, and added to the cell, suchthat expression of the peptide agent is accomplished, e.g., PCTUS97/01019. Regulatable gene therapy systems can also be used. Once themodulator has been administered to the cells, the cells are washed ifdesired and are allowed to incubate under preferably physiologicalconditions for some period. The cells are then harvested and a new geneexpression profile is generated. Thus, e.g., cancer tissue is screenedfor agents that modulate, e.g., induce or suppress, the cancerphenotype. A change in at least one gene, preferably many, of theexpression profile indicates that the agent has an effect on canceractivity. Similarly, altering a biological function or a signalingpathway is indicative of modulator activity. By defining such asignature for the cancer phenotype, screens for new drugs that alter thephenotype are devised. With this approach, the drug target need not beknown and need not be represented in the original gene/proteinexpression screening platform, nor does the level of transcript for thetarget protein need to change. The modulator inhibiting function willserve as a surrogate marker

As outlined above, screens are done to assess genes or gene products.That is, having identified a particular differentially expressed gene asimportant in a particular state, screening of modulators of either theexpression of the gene or the gene product itself is performed.

Use of Modulators to Affect Peptides of the Invention

Measurements of cancer polypeptide activity, or of the cancer phenotypeare performed using a variety of assays. For example, the effects ofmodulators upon the function of a cancer polypeptide(s) are measured byexamining parameters described above. A physiological change thataffects activity is used to assess the influence of a test compound onthe polypeptides of this invention. When the functional outcomes aredetermined using intact cells or animals, a variety of effects can beassesses such as, in the case of a cancer associated with solid tumors,tumor growth, tumor metastasis, neovascularization, hormone release,transcriptional changes to both known and uncharacterized geneticmarkers (e.g., by Northern blots), changes in cell metabolism such ascell growth or pH changes, and changes in intracellular secondmessengers such as cGNIP.

Methods of Identifying Characterizing Cancer-Associated Sequences

Expression of various gene sequences is correlated with cancer.Accordingly, disorders based on mutant or variant cancer genes aredetermined. In one embodiment, the invention provides methods foridentifying cells containing variant cancer genes, e.g., determining thepresence of, all or part, the sequence of at least one endogenous cancergene in a cell. This is accomplished using any number of sequencingtechniques. The invention comprises methods of identifying the cancergenotype of an individual, e.g., determining all or part of the sequenceof at least one gene of the invention in the individual. This isgenerally done in at least one tissue of the individual, e.g., a tissueset forth in Table I, and may include the evaluation of a number oftissues or different samples of the same tissue. The method may includecomparing the sequence of the sequenced gene to a known cancer gene,i.e., a wild-type gene to determine the presence of family members,homologies, mutations or variants. The sequence of all or part of thegene can then be compared to the sequence of a known cancer gene todetermine if any differences exist. This is done using any number ofknown homology programs, such as BLAST, Bestfit, etc. The presence of adifference in the sequence between the cancer gene of the patient andthe known cancer gene correlates with a disease state or a propensityfor a disease state, as outlined herein.

In a preferred embodiment, the cancer genes are used as probes todetermine the number of copies of the cancer gene in the genome. Thecancer genes are used as probes to determine the chromosomallocalization of the cancer genes. Information such as chromosomallocalization finds use in providing a diagnosis or prognosis inparticular when chromosomal abnormalities such as translocations, andthe like are identified in the cancer gene locus.

XIV.) KITS/ARTICLES OF MANUFACTURE

For use in the diagnostic and therapeutic applications described herein,kits are also within the scope of the invention. Such kits can comprisea carrier, package or container that is compartmentalized to receive oneor more containers such as vials, tubes, and the like, each of thecontainer(s) comprising one of the separate elements to be used in themethod. For example, the container(s) can comprise a probe that is orcan be detectably labeled. Such probe can be an antibody orpolynucleotide specific for a FIG. 2-related protein or a FIG. 2 gene ormessage, respectively. Where the method utilizes nucleic acidhybridization to detect the target nucleic acid, the kit can also havecontainers containing nucleotide(s) for amplification of the targetnucleic acid sequence and/or a container comprising a reporter-means,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, florescent, orradioisotope label. The kit can include all or part of the amino acidsequences in FIG. 2 or FIG. 3 or analogs thereof, or a nucleic acidmolecules that encodes such amino acid sequences.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes; carrier, package, container, vial and/ortube labels listing contents and/or instructions for use, and packageinserts with instructions for use.

A label can be present on the container to indicate that the compositionis used for a specific therapy or non-therapeutic application, such as adiagnostic or laboratory application, and can also indicate directionsfor either in vivo or in vitro use, such as those described herein.Directions and or other information can also be included on an insert(s)or label(s) which is included with or on the kit.

The terms “kit” and “article of manufacture” can be used as synonyms.

In another embodiment of the invention, an article(s) of manufacturecontaining compositions, such as amino acid sequence(s), smallmolecule(s), nucleic acid sequence(s), and/or antibody(s), e.g.,materials useful for the diagnosis, prognosis, prophylaxis and/ortreatment of neoplasias of tissues such as those set forth in Table I isprovided. The article of manufacture typically comprises at least onecontainer and at least one label. Suitable containers include, forexample, bottles, vials, syringes, and test tubes. The containers can beformed from a variety of materials such as glass or plastic. Thecontainer can hold amino acid sequence(s), small molecule(s), nucleicacid sequence(s), and/or antibody(s), in one embodiment the containerholds a polynucleotide for use in examining the mRNA expression profileof a cell, together with reagents used for this purpose.

The container can alternatively hold a composition which is effectivefor treating, diagnosis, prognosing or prophylaxing a condition and canhave a sterile access port (for example the container can be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). The active agents in the composition canbe an antibody capable of specifically binding 24P4C12 and modulatingthe function of 24P4C12.

The label can be on or associated with the container. A label a can beon a container when letters, numbers or other characters forming thelabel are molded or etched into the container itself; a label can beassociated with a container when it is present within a receptacle orcarrier that also holds the container, e.g., as a package insert. Thelabel can indicate that the composition is used for diagnosing,treating, prophylaxing or prognosing a condition, such as a neoplasia ofa tissue set forth in Table I. The article of manufacture can furthercomprise a second container comprising a pharmaceutically-acceptablebuffer, such as phosphate-buffered saline, Ringer's solution and/ordextrose solution. It can further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, stirrers, needles, syringes, and/or package inserts withindications and/or instructions for use.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples that follow, none of which are intendedto limit the scope of the invention.

Example 1 SSH-Generated Isolation of cDNA Fragment of the 24P4C12 Gene

Suppression Subtractive Hybridization (SSH) was used to identify cDNAscorresponding to genes that may be differentially expressed in prostatecancer. The SSH reaction utilized cDNA from the LAPC-9 AD prostatecancer xenograft. The gene 24P4C12 was derived from an LAPC-9 AD minusbenign prostatic hyperplasia experiment.

The 24P4C12 SSH cDNA of 160 bp is listed in FIG. 1. The full length24P4C12 cDNAs and ORFs are described in FIG. 2 with the proteinsequences listed in FIG. 3.

Materials and Methods

Human Tissues:

The patient cancer and normal tissues were purchased from differentsources such as the NDRI (Philadelphia, Pa.). mRNA for some normaltissues were purchased from Clontech, Palo Alto, Calif.

RNA Isolation:

Tissues were homogenized in Trizol reagent (Life Technologies, GibcoBRL) using 10 ml/g tissue isolate total RNA. Poly A RNA was purifiedfrom total RNA using Qiagen's Oligotex mRNA Mini and Midi kits. Totaland mRNA were quantified by spectrophotometric analysis (O.D. 260/280nm) and analyzed by gel electrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used.

DPNCDN (cDNA Synthesis Primer):

5′TTTTGATCAAGCTT₃₀3′ (SEQ ID NO: 33)

Adaptor 1:

(SEQ ID NO: 34) 5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ (SEQ IDNO: 35) 3′GGCCCGTCCTAG5′

Adaptor 2:

(SEQ ID NO: 36) 5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NO:37) 3′CGGCTCCTAG5′

PCR Primer 1:

5′CTAATACGACTCACTATAGGGC3′ (SEQ ID NO: 38)

Nested Primer (NP)1:

5′TCGAGCGGCCGCCCGGGCAGGA3′ (SEQ ID NO: 39)

Nested Primer (NP)2:

5′AGCGTGGTCGCGGCCGAGGA3′ (SEQ ID NO: 40)

Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNAscorresponding to genes that may be differentially expressed in prostatecancer. The SSH reaction utilized cDNA from prostate cancer and normaltissues.

The gene 24P4C12 sequence was derived from LAPC-4AD prostate cancerxenograft minus begnin prostatic hyperplasia cDNA subtraction. The SSHDNA sequence (FIG. 1) was identified.

The cDNA derived from a pool of normal tissues and benign prostatichyperplasia was used as the source of the “driver” cDNA, while the cDNAfrom LAPC-4AD xenograft was used as the source of the “tester” cDNA.Double stranded cDNAs corresponding to tester and driver cDNAs weresynthesized from 2 μg of poly(A)⁺ RNA isolated from the relevantxenograft tissue, as described above, using CLONTECH's PCR-Select cDNASubtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- andsecond-strand synthesis were carried out as described in the Kit's usermanual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1).The resulting cDNA was digested with Dpn II for 3 hrs at 37° C. DigestedcDNA was extracted with phenol/chloroform (1:1) and ethanolprecipitated.

Driver cDNA was generated by combining in a 1:1 ratio Dpn II digestedcDNA from the relevant tissue source (see above) with a mix of digestedcDNAs derived from the nine normal tissues: stomach, skeletal muscle,lung, brain, liver, kidney, pancreas, small intestine, and heart.

Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA fromthe relevant tissue source (see above) (400 ng) in 5 μl of water. Thediluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 andAdaptor 2 (10 μM), in separate ligation reactions, in a total volume of10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH).Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C.for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) ofdriver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1-and Adaptor 2-ligated tester cDNA. In a final volume of 4 pt, thesamples were overlaid with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generatedfrom SSH:

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, and 72° C. for 1.5 minutes. The PCR productswere analyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid culture overnight. To identifyinserts, PCR amplification was performed on 1 ul of bacterial cultureusing the conditions of PCR1 and NP1 and NP2 as primers. PCR productswere analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis:

First strand cDNAs can be generated from 1 μg of mRNA with oligo (dT)12-18 priming using the Gibco-BRL Superscript Preamplification system.The manufacturer's protocol was used which included an incubation for 50min at 42° C. with reverse transcriptase followed by RNAse H treatmentat 37° C. for 20 min. After completing the reaction, the volume can beincreased to 200 μl with water prior to normalization. First strandcDNAs from 16 different normal human tissues can be obtained fromClontech.

Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO:41) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 42) to amplifyβ-actin. First strand cDNA (5 μl) were amplified in a total volume of 50μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech,10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH8.3) and 1× Klentaq DNApolymerase (Clontech). Five μl of the PCR reaction can be removed at 18,20, and 22 cycles and used for agarose gel electrophoresis. PCR wasperformed using an MJ Research thermal cycler under the followingconditions: Initial denaturation can be at 94° C. for 15 sec, followedby a 18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C.for 5 sec. A final extension at 72° C. was carried out for 2 min. Afteragarose gel electrophoresis, the band intensities of the 283 b.p.β-actin bands from multiple tissues were compared by visual inspection.Dilution factors for the first strand cDNAs were calculated to result inequal β-actin band intensities in all tissues after 22 cycles of PCR.Three rounds of normalization can be required to achieve equal bandintensities in all tissues after 22 cycles of PCR.

To determine expression levels of the 24P4C12 gene, 5 μl of normalizedfirst strand cDNA were analyzed by PCR using 26, and 30 cycles ofamplification. Semi-quantitative expression analysis can be achieved bycomparing the PCR products at cycle numbers that give light bandintensities. The primers used for RT-PCR were designed using the 24P4C12SSH sequence and are listed below:

24P4C12.1 5′-AGATGAGGAGGAGGACAAAGGTG-3′ (SEQ ID NO: 43) 24P4C12.2 5′-ACTGCTGGGAGGAGTACCGAGTG-3′ (SEQ ID NO: 44)

Example 2 Isolation of Full Length 24P4C12 Encoding cDNA

The 24P4C12 SSH cDNA sequence was derived from a substraction consistingof LAPC-4AD xenograft minus benign prostatic hyperplasia. The SSH cDNAsequence (FIG. 1) was designated 24P4C12.

The isolated gene fragment of 160 bp encodes a putative open readingframe (ORF) of 53 amino acids and exhibits significant homology to anEST derived from a colon tumor library. Two larger cDNA clones wereobtained by gene trapper experiments, GTE9 and GTF8. The ORF revealed asignificant homology to the mouse gene NG22 and the C. elegans geneCEESB82F. NG22 was recently identified as one of many ORFs within agenomic BAC clone that encompasses the MHC class III in the mousegenome. Both NG22 and CEESB82F appear to be genes that contain 12transmembrane domains. This suggests that the gene encoding 24P4C12contains 12 transmembrane domains and is the human homologue of mouseNG22 and C. elegans CEESB82F. Functional studies in Ce. elegans mayreveal the biological role of these homologs. If 24P4C12 is a cellsurface marker, then it may have an application as a potential imagingreagent and/or therapeutic target in prostate cancer.

The 24P4C12 v.1 of 2587 bp codes for a protein of 710 amino acids (FIG.2 and FIG. 3). Other variants of 24P4C12 were also identified and theseare listed in FIGS. 2 and 3. 24P4C12 v.1, v.3, v.5 and v.6 proteins are710 amino acids in length and differ from each other by one amino acidas shown in FIGS. 11. 24P4C12 v.2 and v.4 code for the same protein as24P4C12 v.1. 24P4C12 v.7, v.8 and v.9 are alternative splice variantsand code for proteins of 598, 722 and 712 amino acids in length,respectively.

Example 3 Chromosomal Mapping of 24P4C12

Chromosomal localization can implicate genes in disease pathogenesis.Several chromosome mapping approaches are available includingfluorescent in situ hybridization (FISH), human/hamster radiation hybrid(RH) panels (Walter et al., 1994; Nature Genetics 7:22; ResearchGenetics, Huntsville Ala.), human-rodent somatic cell hybrid panels suchas is available from the Coriell Institute (Camden, N.J.), and genomicviewers utilizing BLAST homologies to sequenced and mapped genomicclones (NCBI, Bethesda, Md.). 24P4C12 maps to chromosome 6p21.3 using24P4C12 sequence and the NCBI BLAST tool located on the World Wide Webat (.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG=Hs).

Example 4 Expression Analysis of 24P4C12

Expression analysis by RT-PCR demonstrated that 24P4C12 is stronglyexpressed in prostate and ovary cancer patient specimens (FIG. 14).First strand cDNA was generated from vital pool 1 (kidney, liver andlung), vital pool 2 (colon, pancreas and stomach), a pool of prostatecancer xenografts (LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-9AI), prostatecancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool,ovary cancer pool, breast cancer pool, and cancer metastasis pool.Normalization was performed by PCR using primers to actin.Semi-quantitative PCR, using primers to 24P4C12, was performed at 26 and30 cycles of amplification. Results show strong expression of 24P4C12 inprostate cancer pool and ovary cancer pool. Expression was also detectedin prostate cancer xenografts, bladder cancer pool, kidney cancer pool,colon cancer pool, breast cancer pool, cancer metastasis pool, vitalpool 1, and vital pool 2.

Extensive northern blot analysis of 24P4C12 in multiple human normaltissues is shown in FIG. 15. Two multiple tissue northern blots(Clontech) both with 2 μg of mRNA/lane were probed with the 24P4C12 SSHsequence. Expression of 24P4C12 was detected in prostate, kidney andcolon. Lower expression is detected in pancreas, lung and placentaamongst all 16 normal tissues tested.

Expression of 24P4C12 was tested in prostate cancer xenografts and celllines. RNA was extracted from a panel of cell lines and prostate cancerxenografts (PrEC, LAPC-4AD, LAPC-4AI, LAPC-9AD, LAPC-9AI, LNCaP, PC-3,DU145, TsuPr, and LAPC-4CL). Northern blot with 10 μg of total RNA/lanewas probed with 24P4C12 SSH sequence. Size standards in kilobases (kb)are indicated on the side. The 24P4C12 transcript was detected inLAPC-4AD, LAPC-4AI, LAPC-9AD, LAPC-9AI, LNCaP, and LAPC-4 CL

Expression of 24P4C12 in patient cancer specimens and human normaltissues is shown in FIG. 16. RNA was extracted from a pool of prostatecancer specimens, bladder cancer specimens, colon cancer specimens,ovary cancer specimens, breast cancer specimens and cancer metastasisspecimens, as well as from normal prostate (NP), normal bladder (NB),normal kidney (NK), and normal colon (NC). Northern blot with 10 μg oftotal RNA/lane was probed with 24P4C12 SSH sequence. Size standards inkilobases (kb) are indicated on the side. Strong expression of 24P4C12transcript was detected in the patient cancer pool specimens, and innormal prostate but not in the other normal tissues tested.

Expression of 24P4C12 was also detected in individual prostate cancerpatient specimens (FIG. 17). RNA was extracted from normal prostate (N),prostate cancer patient tumors (T) and their matched normal adjacenttissues (Nat). Northern blots with 10 μg of total RNA were probed withthe 24P4C12 SSH fragment. Size standards in kilobases are on the side.Results show expression of 24P4C12 in normal prostate and all prostatepatient tumors tested.

Expression of 24P4C12 in colon cancer patient specimens is shown in FIG.18. RNA was extracted from colon cancer cell lines (CL: Colo 205, LoVo,and SK—CO—), normal colon (N), colon cancer patient tumors (T) and theirmatched normal adjacent tissues (Nat). Northern blots with 10 μg oftotal RNA were probed with the 24P4C12 SSH fragment. Size standards inkilobases are on the side. Results show expression of 24P4C12 in normalcolon and all colon patient tumors tested. Expression was detected inthe cell lines Colo 205 and SK—CO—, but not in LoVo.

FIG. 20 displays expression results of 24P4C12 in lung cancer patientspecimens. Ma was extracted from lung cancer cell lines (CL: CALU-1,A427, NCI-H82, NCI-H146), normal lung (N), lung cancer patient tumors(T) and their matched normal adjacent tissues (Nat). Northern blots with10 μg of total RNA were probed with the 24P4C12 SSH fragment. Sizestandards in kilobases are on the side. Results show expression of24P4C12 in lung patient tumors tested, but not in normal lung.Expression was also detected in CALU-1, but not in the other cell linesA427, NCI-H82, and NCI-H₁₄₆.

24P4C12 was assayed in a panel of human stomach and breast cancers (T)and their respective matched normal tissues (N) on RNA dot blots.24P4C12 expression was seen in both stomach and breast cancers. Theexpression detected in normal adjacent tissues (isolated from diseasedtissues) but not in normal tissues (isolated from healthy donors) mayindicate that these tissues are not fully normal and that 24P4C12 may beexpressed in early stage tumors.

The level of expression of 24P4C12 was analyzed and quantitated in apanel of patient cancer tissues. First strand cDNA was prepared from apanel of ovary patient cancer specimens (A), uterus patient cancerspecimens (B), prostate cancer specimens (C), bladder cancer patientspecimens (D), lung cancer patient specimens (E), pancreas cancerpatient specimens (F), colon cancer specimens (G), and kidney cancerspecimens (H). Normalization was performed by PCR using primers toactin. Semi-quantitative PCR, using primers to 24P4C12, was performed at26 and 30 cycles of amplification. Samples were run on an agarose gel,and PCR products were quantitated using the AlphaImager software.Expression was recorded as absent, low, medium or strong. Results showexpression of 24P4C12 in the majority of patient cancer specimenstested, 73.3% of ovary patient cancer specimens, 83.3% of uterus patientcancer specimens, 95.0% of prostate cancer specimens, 61.1% of bladdercancer patient specimens, 80.6% of lung cancer patient specimens, 87.5%of pancreas cancer patient specimens, 87.5% of colon cancer specimens,68.4% of clear cell renal carcinoma, 100% of papillary renal cellcarcinoma. The restricted expression of 24P4C12 in normal tissues andthe expression detected in prostate cancer, ovary cancer, bladdercancer, colon cancer, lung cancer pancreas cancer, uterus cancer, kidneycancer, stomach cancer and breast cancer suggest that 24P4C12 is apotential therapeutic target and a diagnostic marker for human cancers.

Example 5 Transcript Variants of 24P4C12

Transcript variants are variants of mature mRNA from the same gene whicharise by alternative transcription or alternative splicing. Alternativetranscripts are transcripts from the same gene but start transcriptionat different points. Splice variants are mRNA variants spliceddifferently from the same transcript. In eukaryotes, when a multi-exongene is transcribed from genomic DNA, the initial RNA is spliced toproduce functional mRNA, which has only exons and is used fortranslation into an amino acid sequence. Accordingly, a given gene canhave zero to many alternative transcripts and each transcript can havezero to many splice variants. Each transcript variant has a unique exonmakeup, and can have different coding and/or non-coding (5′ or 3′ end)portions, from the original transcript. Transcript variants can code forsimilar or different proteins with the same or a similar function or canencode proteins with different functions, and can be expressed in thesame tissue at the same time, or in different tissues at the same time,or in the same tissue at different times, or in different tissues atdifferent times. Proteins encoded by transcript variants can havesimilar or different cellular or extracellular localizations, e.g.,secreted versus intracellular.

Transcript variants are identified by a variety of art-accepted methods.For example, alternative transcripts and splice variants are identifiedby full-length cloning experiment, or by use of full-length transcriptand EST sequences. First, all human ESTs were grouped into clusterswhich show direct or indirect identity with each other. Second, ESTs inthe same cluster were further grouped into sub-clusters and assembledinto a consensus sequence. The original gene sequence is compared to theconsensus sequence(s) or other full-length sequences. Each consensussequence is a potential splice variant for that gene. Even when avariant is identified that is not a full-length clone, that portion ofthe variant is very useful for antigen generation and for furthercloning of the full-length splice variant, using techniques known in theart.

Moreover, computer programs are available in the art that identifytranscript variants based on genomic sequences. Genomic-based transcriptvariant identification programs include FgenesH (A. Salamov and V.Solovyev, “Ab initio gene finding in Drosophila genomic DNA,” GenomeResearch. 2000 April; 10(4):516-22); Grail (URL atcompbio.ornl.gov/Grail-bin/EmptyGrailForm) and GenScan (URL atgenes.mit.edu/GENSCAN.html). For a general discussion of splice variantidentification protocols see., e.g., Southan, C., A genomic perspectiveon human proteases, FEBS Lett. 2001 Jun. 8; 498(2-3):214-8; de Souza, S.J., et al., Identification of human chromosome 22 transcribed sequenceswith ORF expressed sequence tags, Proc. Natl Acad Sci USA. 2000 Nov. 7;97(23):12690-3.

To further confirm the parameters of a transcript variant, a variety oftechniques are available in the art, such as full-length cloning,proteomic validation, PCR-based validation, and 5′ RACE validation, etc.(see e.g., Proteomic Validation: Brennan, S. O., et al., Albumin bankspeninsula: a new termination variant characterized by electrospray massspectrometry, Biochem Biophys Acta. 1999 Aug. 17; 1433(1-2):321-6;Ferranti P, et al., Differential splicing of pre-messenger RNA producesmultiple forms of mature caprine alpha(s1)-casein, Eur J Biochem. 1997Oct. 1; 249(1):1-7. For PCR-based Validation: Wellmann S, et al.,Specific reverse transcription-PCR quantification of vascularendothelial growth factor (VEGF) splice variants by LightCyclertechnology, Clin Chem. 2001 April; 47(4):654-60; Jia, H. P., et al.,Discovery of new human beta-defensins using a genomics-based approach,Gene. 2001 Jan. 24; 263(1-2):211-8. For PCR-based and 5′ RACEValidation: Brigle, K. E., et al., Organization of the murine reducedfolate carrier gene and identification of variant splice forms, BiochemBiophys Acta. 1997 Aug. 7; 1353(2): 191-8).

It is known in the art that genomic regions are modulated in cancers.When the genomic region to which a gene maps is modulated in aparticular cancer, the alternative transcripts or splice variants of thegene are modulated as well. Disclosed herein is that 24P4C12 has aparticular expression profile related to cancer. Alternative transcriptsand splice variants of 24P4C12 may also be involved in cancers in thesame or different tissues, thus serving as tumor-associatedmarkers/antigens.

The exon composition of the original transcript, designated as 24P4C12v.1, is shown in Table LI. Using the full-length gene and EST sequences,three transcript variants were identified, designated as 24P4C12 v.7,v.8 and v.9. Compared with 24P4C12 v.1, transcript variant 24P4C12 v.7has spliced out exons 10 and 11 from variant 24P4C12 v.1, as shown inFIG. 12. Variant 24P4C12 v.8 inserted 36 bp in between 1931 and 1932 ofvariant 24P4C12 v.1 and variant 24P4C12 v.9 replaced with 36 bp thesegment 1136-1163 of variant 24P4C12 v.1. Theoretically, each differentcombination of exons in spatial order, e.g. exons 2 and 3, is apotential splice variant. FIG. 12 shows the schematic alignment of exonsof the four transcript variants.

Tables LII through LXIII are set forth on a variant by variant basis.Tables LII, LVI, and LX show nucleotide sequences of the transcriptvariant. Tables LIII, LVII, and LXI show the alignment of the transcriptvariant with the nucleic acid sequence of 24P4C12 v.1. Tables LIV,LVIII, and LXII lay out the amino acid translation of the transcriptvariant for the identified reading frame orientation. Tables LV, LIX,and LXIII display alignments of the amino acid sequence encoded by thesplice variant with that of 24P4C12 v.1.

Example 6 Single Nucleotide Polymorphisms of 24P4C12

A Single Nucleotide Polymorphism (SNP) is a single base pair variationin a nucleotide sequence at a specific location. At any given point ofthe genome, there are four possible nucleotide base pairs: AIT, C/G, G/Cand T/A. Genotype refers to the specific base pair sequence of one ormore locations in the genome of an individual. Haplotype refers to thebase pair sequence of more than one location on the same DNA molecule(or the same chromosome in higher organisms), often in the context ofone gene or in the context of several tightly linked genes. SNPs thatoccur on a cDNA are called cSNPs. These cSNPs may change amino acids ofthe protein encoded by the gene and thus change the functions of theprotein. Some SNPs cause inherited diseases; others contribute toquantitative variations in phenotype and reactions to environmentalfactors including diet and drugs among individuals. Therefore, SNPsand/or combinations of alleles (called haplotypes) have manyapplications, including diagnosis of inherited diseases, determinationof drug reactions and dosage, identification of genes responsible fordiseases, and analysis of the genetic relationship between individuals(P. Nowotny, J. M. Kwon and A. M. Goate, “SNP analysis to dissect humantraits,” Curr. Opin. Neurobiol. 2001 October; 11(5):637-641; M.Pirmohamed and B. K. Park, “Genetic susceptibility to adverse drugreactions,” Trends Pharmacol. Sci. 2001 June; 22(6):298-305; J. H.Riley, C. J. Allan, E. Lai and A. Roses, “The use of single nucleotidepolymorphisms in the isolation of common disease genes,”Pharmacogenomics. 2000 February; 1(1):39-47; R. Judson, J. C. Stephensand A. Windemuth, “The predictive power of haplotypes in clinicalresponse,” Pharmacogenomics. 2000 feb; 1(1):15-26).

SNPs are identified by a variety of art-accepted methods (P. Bean, “Thepromising voyage of SNP target discovery,” Am. Clin. Lab. 2001October-November; 20(9):18-20; K. M. Weiss, “In search of humanvariation,” Genome Res. 1998 July; 8(7):691-697; M. M. She, “Enablinglarge-scale pharmacogenetic studies by high-throughput mutationdetection and genotyping technologies,” Clin. Chem. 2001 February;47(2):164-172). For example, SNPs are identified by sequencing DNAfragments that show polymorphism by gel-based methods such asrestriction fragment length polymorphism (RFLP) and denaturing gradientgel electrophoresis (DGGE). They can also be discovered by directsequencing of DNA samples pooled from different individuals or bycomparing sequences from different DNA samples. With the rapidaccumulation of sequence data in public and private databases, one candiscover SNPs by comparing sequences using computer programs (Z. Gu, L.Hillier and P. Y. Kwok, “Single nucleotide polymorphism hunting incyberspace,” Hum. Mutat. 1998; 12(4):221-225). SNPs can be verified andgenotype or haplotype of an individual can be determined by a variety ofmethods including direct sequencing and high throughput microarrays (P.Y. Kwok, “Methods for genotyping single nucleotide polymorphisms,” Annu.Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K.Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft,“High-throughput SNP genotyping with the Masscode system,” Mol. Diagn.2000 December; 5(4):329-340).

Using the methods described above, five SNPs were identified in theoriginal transcript, 24P4C12 v.1, at positions 542 (G/A), 564 (G/A), 818(C/T), 981 (A/G) and 1312 (A/C). The transcripts or proteins withalternative alleles were designated as variants 24P4C12 v.2, v.3, v.4,v.5 and v.6, respectively. FIG. 10 shows the schematic alignment of theSNP variants. FIG. 11 shows the schematic alignment of protein variants,corresponding to nucleotide variants. Nucleotide variants that code forthe same amino acid sequence as variant 1 are not shown in FIG. 11.These alleles of the SNPs, though shown separately here, can occur indifferent combinations (haplotypes) and in any one of the transcriptvariants (such as 24P4C12 v.7) that contains the sequence context of theSNPs.

Example 7 Production of Recombinant 24P4C12 in Prokaryotic Systems

To express recombinant 24P4C12 and 24P4C12 variants in prokaryoticcells, the full or partial length 24P4C12 and 24P4C12 variant cDNAsequences are cloned into any one of a variety of expression vectorsknown in the art. The full length cDNA, or any 8, 9, 10, 11, 12,13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or morecontiguous amino acids from 24P4C12, variants, or analogs thereof areused.

A. In vitro transcription and translation constructs:

PCRII: To generate 24P4C12 sense and anti-sense RNA probes for RNA insitu investigations, pCRII constructs (Invitrogen, Carlsbad Calif.) aregenerated encoding either all or fragments of the 24P4C12 cDNA. ThepCRII vector has Sp6 and T7 promoters flanking the insert to drive thetranscription of 24P4C12 RNA for use as probes in RNA in situhybridization experiments. These probes are used to analyze the cell andtissue expression of 24P4C12 at the RNA level. Transcribed 24P4C12 RNArepresenting the cDNA amino acid coding region of the 24P4C12 gene isused in in vitro translation systems such as the TnT™ CoupledReticulolysate System (Promega, Corp., Madison, Wis.) to synthesize24P4C12 protein.

B. Bacterial Constructs:

pGEX Constructs: To generate recombinant 24P4C12 proteins in bacteriathat are fused to the Glutathione S-transferase (GST) protein, all orparts of the 24P4C12 cDNA or variants are cloned into the GST-fusionvector of the pGEX family (Amersham Pharmacia Biotech, Piscataway,N.J.). These constructs allow controlled expression of recombinant24P4C12 protein sequences with GST fused at the amino-terminus and a sixhistidine epitope (6×His) at the carboxyl-terminus. The GST and 6× Histags permit purification of the recombinant fusion protein from inducedbacteria with the appropriate affinity matrix and allow recognition ofthe fusion protein with anti-GST and anti-His antibodies. The 6× His tagis generated by adding 6 histidine codons to the cloning primer at the3′ end, e.g., of the open reading frame (ORF). A proteolytic cleavagesite, such as the PreScission™ recognition site in pGEX-6P-1, may beemployed such that it permits cleavage of the GST tag from24P4C12-related protein. The ampicillin resistance gene and pBR322origin permits selection and maintenance of the pGEX plasmids in E.coli.

pMAL Constructs: To generate, in bacteria, recombinant 24P4C12 proteinsthat are fused to maltose-binding protein (MBP), all or parts of the24P4C12 cDNA protein coding sequence are fused to the MBP gene bycloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs,Beverly, Mass.). These constructs allow controlled expression ofrecombinant 24P4C12 protein sequences with MBP fused at theamino-terminus and a 6×His epitope tag at the carboxyl-terminus. The MBPand 6×His tags permit purification of the recombinant protein frominduced bacteria with the appropriate affinity matrix and allowrecognition of the fusion protein with anti-MBP and anti-His antibodies.The 6×His epitope tag is generated by adding 6 histidine codons to the3′ cloning primer. A Factor Xa recognition site permits cleavage of thepMAL tag from 24P4C12. The pMAL-c2X and pMAL-p2X vectors are optimizedto express the recombinant protein in the cytoplasm or periplasmrespectively. Periplasm expression enhances folding of proteins withdisulfide bonds.

pET Constructs: To express 24P4C12 in bacterial cells, all or parts ofthe 24P4C12 cDNA protein coding sequence are cloned into the pET familyof vectors (Novagen, Madison, Wis.). These vectors allow tightlycontrolled expression of recombinant 24P4C12 protein in bacteria withand without fusion to proteins that enhance solubility, such as NusA andthioredoxin (Trx), and epitope tags, such as 6×His and S-Tag™ that aidpurification and detection of the recombinant protein. For example,constructs are made utilizing pET NusA fusion system 43.1 such thatregions of the 24P4C12 protein are expressed as amino-terminal fusionsto NusA.

C. Yeast Constructs:

pESC Constructs: To express 24P4C12 in the yeast species Saccharomycescerevisiae for generation of recombinant protein and functional studies,all or parts of the 24P4C12 cDNA protein coding sequence are cloned intothe pESC family of vectors each of which contain 1 of 4 selectablemarkers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La Jolla, Calif.).These vectors allow controlled expression from the same plasmid of up to2 different genes or cloned sequences containing either Flag™ or Mycepitope tags in the same yeast cell. This system is useful to confirmprotein-protein interactions of 24P4C12. In addition, expression inyeast yields similar post-translational modifications, such asglycosylations and phosphorylations, that are found when expressed ineukaryotic cells.

pESP Constructs: To express 24P4C12 in the yeast species Saccharomycespombe, all or parts of the 24P4C12 cDNA protein coding sequence arecloned into the pESP family of vectors. These vectors allow controlledhigh level of expression of a 24P4C12 protein sequence that is fused ateither the amino terminus or at the carboxyl terminus to GST which aidspurification of the recombinant protein. A Flag™ epitope tag allowsdetection of the recombinant protein with anti-Flag™ antibody.

Example 8 Production of Recombinant 24P4C12 in Higher Eukaryotic Systems

A. Mammalian Constructs:

To express recombinant 24P4C12 in eukaryotic cells, the full or partiallength 24P4C12 cDNA sequences can be cloned into any one of a variety ofexpression vectors known in the art. One or more of the followingregions of 24P4C12 are expressed in these constructs, amino acids 1 to710, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from24P4C12 v.1 through v.6; amino acids 1 to 598, or any 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30or more contiguous amino acids from 24P4C12 v.7, amino acids 1 to 722,or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 or more contiguous amino acids from 24P4C12 v.8,amino acids 1 to 712, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous aminoacids from 24P4C12 v.9, variants, or analogs thereof.

The constructs can be transfected into any one of a wide variety ofmammalian cells such as 293T cells. Transfected 293T cell lysates can beprobed with the anti-24P4C12 polyclonal serum, described herein.

pcDNA3.1/MycHis Constructs: To express 24P4C12 in mammalian cells, a24P4C12 ORF, or portions thereof, of 24P4C12 with a consensus Kozaktranslation initiation site was cloned into pcDNA3.1/MycHis Version A(Invitrogen, Carlsbad, Calif.). Protein expression is driven from thecytomegalovirus (CMV) promoter. The recombinant proteins have the mycepitope and 6×His epitope fused to the carboxyl-terminus. ThepcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability, along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheNeomycin resistance gene can be used, as it allows for selection ofmammalian cells expressing the protein and the ampicillin resistancegene and ColE1 origin permits selection and maintenance of the plasmidin E. coli. Figure Art-1 demonstrates expression of 24P4C12 from thepcDNA3.1/MycHis construct in transiently transfected 293T cells.

pcDNA4/HisMax Constructs: To express 24P4C12 in mammalian cells, a24P4C12 ORF, or portions thereof, of 24P4C12 are cloned intopcDNA4/HisMax Version A (Invitrogen, Carlsbad, Calif.). Proteinexpression is driven from the cytomegalovirus (CMV) promoter and theSP16 translational enhancer. The recombinant protein has XpreSS™ and sixhistidine (6×His) epitopes fused to the amino-terminus. ThepcDNA4/HisMax vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheZeocin resistance gene allows for selection of mammalian cellsexpressing the protein and the ampicillin resistance gene and ColE1origin permits selection and maintenance of the plasmid in E. coli.

pcDNA3.1/CT-GFP-TOPO Construct: To express 24P4C12 in mammalian cellsand to allow detection of the recombinant proteins using fluorescence, a24P4C12 ORF, or portions thereof, with a consensus Kozak translationinitiation site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen,Calif.). Protein expression is driven from the cytomegalovirus (CMV)promoter. The recombinant proteins have the Green Fluorescent Protein(GFP) fused to the carboxyl-terminus facilitating non-invasive, in vivodetection and cell biology studies. The pcDNA3.1 CT-GFP-TOPO vector alsocontains the bovine growth hormone (BGH) polyadenylation signal andtranscription termination sequence to enhance mRNA stability along withthe SV40 origin for episomal replication and simple vector rescue incell lines expressing the large T antigen. The Neomycin resistance geneallows for selection of mammalian cells that express the protein and theampicillin resistance gene and ColE1 origin permits selection andmaintenance of the plasmid in E. coli. Additional constructs with anamino-terminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning theentire length of a 24P4C12 protein.

pTag5: A 24P4C12 ORF, or portions thereof, were cloned into pTag-5. Thisvector is similar to pAPtag but without the alkaline phosphatase fusion.This construct generates 24P4C12 protein with an amino-terminal IgGκsignal sequence and myc and 6×His epitope tags at the carboxyl-terminusthat facilitate detection and affinity purification. The resultingrecombinant 24P4C12 protein were optimized for secretion into the mediaof transfected mammalian cells, and is used as immunogen or ligand toidentity proteins such as ligands or receptors that interact with the24P4C12 proteins. Protein expression is driven from the CMV promoter.The Zeocin resistance gene present in the vector allows for selection ofmammalian cells expressing the protein, and the ampicillin resistancegene permits selection of the plasmid in E. coli. Figure Art-3 showsexpression of 24P4C12 from two different pTag5 constructs.

PAPtag: A 24P4C12 ORF, or portions thereof, is cloned into pAPtag-5(GenHunter Corp. Nashville, Tenn.). This construct generates an alkalinephosphatase fusion at the carboxyl-terminus of a 24P4C12 protein whilefusing the IgGκ signal sequence to the amino-terminus. Constructs arealso generated in which alkaline phosphatase with an amino-terminal IgGκsignal sequence is fused to the amino-terminus of a 24P4C12 protein. Theresulting recombinant 24P4C12 proteins are optimized for secretion intothe media of transfected mammalian cells and can be used to identityproteins such as ligands or receptors that interact with 24P4C12proteins. Protein expression is driven from the CMV promoter and therecombinant proteins also contain myc and 6×His epitopes fused at thecarboxyl-terminus that facilitates detection and purification. TheZeocin resistance gene present in the vector allows for selection ofmammalian cells expressing the recombinant protein and the ampicillinresistance gene permits selection of the plasmid in E. coli.

PsecFc: A 24P4C12 ORF, or portions thereof, is also cloned into psecFc.The psecFc vector was assembled by cloning the human immunoglobulin G1(IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen,California). This construct generates an IgG1 Fc fusion at thecarboxyl-terminus of the 24P4C12 proteins, while fusing the IgGK signalsequence to N-terminus. 24P4C12 fusions utilizing the murine IgG1 Fcregion are also used. The resulting recombinant 24P4C12 proteins areoptimized for secretion into the media of transfected mammalian cells,and can be used as immunogens or to identify proteins such as ligands orreceptors that interact with 24P4C12 protein. Protein expression isdriven from the CMV promoter. The hygromycin resistance gene present inthe vector allows for selection of mammalian cells that express therecombinant protein, and the ampicillin resistance gene permitsselection of the plasmid in E. coli.

pSRα Constructs: To generate mammalian cell lines that express 24P4C12constitutively, 24P4C12 ORF, or portions thereof, of 24P4C12 were clonedinto pSRα constructs. Amphotropic and ecotropic retroviruses weregenerated by transfection of pSRα constructs into the 293T-10A1packaging line or co-transfection of pSRα and a helper plasmid(containing deleted packaging sequences) into the 293 cells,respectively. The retrovirus is used to infect a variety of mammaliancell lines, resulting in the integration of the cloned gene, 24P4C12,into the host cell-lines. Protein expression is driven from a longterminal repeat (LTR). The Neomycin resistance gene present in thevector allows for selection of mammalian cells that express the protein,and the ampicillin resistance gene and ColE1 origin permit selection andmaintenance of the plasmid in E. coli. The retroviral vectors canthereafter be used for infection and generation of various cell linesusing, for example, PC3, NIH 3T3, TsuPr1, 293 or rat-1 cells. FIG. 23shows RNA expression of 24P4C12 driven from the 24P4Ct2.pSRα constructin stably transduced PC3, 3T3 and 300.19 cells. Figure Art-2 shows24P4C12 protein expression in PC3 cells stably transduced with24P4C12.pSRα construct.

Additional pSRα constructs are made that fuse an epitope tag such as theFLAGT™ tag to the carboxyl-terminus of 24P4C12 sequences to allowdetection using anti-Flag antibodies. For example, the FLAG™ sequence 5′gat tac aag gat gac gac gat aag 3′ (SEQ ID NO: 45) is added to cloningprimer at the 3′ end of the ORF. Additional pSRα constructs are made toproduce both amino-terminal and carboxyl-terminal GFP and myc/6×Hisfusion proteins of the full-length 24P4C12 proteins.

Additional Viral Vectors: Additional constructs are made forviral-mediated delivery and expression of 24P4C12. High virus titerleading to high level expression of 24P4C12 is achieved in viraldelivery systems such as adenoviral vectors and herpes amplicon vectors.A 24P4C12 coding sequences or fragments thereof are amplified by PCR andsubcloned into the AdEasy shuttle vector (Stratagene). Recombination andvirus packaging are performed according to the manufacturer'sinstructions to generate adenoviral vectors. Alternatively, 24P4C12coding sequences or fragments thereof are cloned into the HSV-1 vector(Imgenex) to generate herpes viral vectors. The viral vectors arethereafter used for infection of various cell lines such as PC3, NIH3T3, 293 or rat-1 cells.

Regulated Expression Systems: To control expression of 24P4C12 inmammalian cells, coding sequences of 24P4C12, or portions thereof, arecloned into regulated mammalian expression systems such as the T-RexSystem (Invitrogen), the GeneSwitch System (Invitrogen) and thetightly-regulated Ecdysone System (Sratagene). These systems allow thestudy of the temporal and concentration dependent effects of recombinant24P4C12. These vectors are thereafter used to control expression of24P4C12 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

B. Baculovirus Expression Systems

To generate recombinant 24P4C12 proteins in a baculovirus expressionsystem, 24P4C12 ORF, or portions thereof, are cloned into thebaculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides aHis-tag at the N-terminus. Specifically, pBlueBac-24P4C12 isco-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9(Spodoptera frugiperda) insect cells to generate recombinant baculovirus(see Invitrogen instruction manual for details). Baculovirus is thencollected from cell supernatant and purified by plaque assay.

Recombinant 24P4C12 protein is then generated by infection of HighFiveinsect cells (Invitrogen) with purified baculovirus. Recombinant 24P4C12protein can be detected using anti-24P4C12 or anti-His-tag antibody.24P4C12 protein can be purified and used in various cell-based assays oras immunogen to generate polyclonal and monoclonal antibodies specificfor 24P4C12.

Example 9 Antigenicity Profiles and Secondary Structure

FIGS. 5-9 depict graphically five amino acid profiles of the 24P4C12variant 1, assessment available by accessing the ProtScale websitelocated on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) onthe ExPasy molecular biology server.

These profiles: FIG. 5, Hydrophilicity, (Hopp T. P., Woods K. R., 1981.Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 6, Hydropathicity,(Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132); FIG. 7,Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492); FIG.8, Average Flexibility, (Bhaskaran R., and Ponnuswamy P. K., 1988. Int.J. Pept. Protein Res. 32:242-255); FIG. 9, Beta-turn (Deleage, G., RouxB. 1987 Protein Engineering 1:289-294); and optionally others availablein the art, such as on the ProtScale website, were used to identifyantigenic regions of the 24P4C12 protein. Each of the above amino acidprofiles of 24P4C12 were generated using the following ProtScaleparameters for analysis: 1) A window size of 9; 2) 100% weight of thewindow edges compared to the window center; and, 3) amino acid profilevalues normalized to lie between 0 and 1.

Hydrophilicity (FIG. 5), Hydropathicity (FIG. 6) and PercentageAccessible Residues (FIG. 7) profiles were used to determine stretchesof hydrophilic amino acids (i.e., values greater than 0.5 on theHydrophilicity and Percentage Accessible Residues profile, and valuesless than 0.5 on the Hydropathicity profile). Such regions are likely tobe exposed to the aqueous environment, be present on the surface of theprotein, and thus available for immune recognition, such as byantibodies.

Average Flexibility (FIG. 8) and Beta-turn (FIG. 9) profiles determinestretches of amino acids (i.e., values greater than 0.5 on the Beta-turnprofile and the Average Flexibility profile) that are not constrained insecondary structures such as beta sheets and alpha helices. Such regionsare also more likely to be exposed on the protein and thus accessible toimmune recognition, such as by antibodies.

Antigenic sequences of the 24P4C12 protein and of the variant proteinsindicated, e.g., by the profiles set forth in FIG. 5, FIG. 6, FIG. 7,FIG. 8, and/or FIG. 9 are used to prepare immunogens, either peptides ornucleic acids that encode them, to generate therapeutic and diagnosticanti-24P4C12 antibodies. The immunogen can be any 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45,50 or more than 50 contiguous amino acids, or the corresponding nucleicacids that encode them, from the 24P4C12 protein variants listed inFIGS. 2 and 3. In particular, peptide immunogens of the invention cancomprise, a peptide region of at least 5 amino acids of FIGS. 2 and 3 inany whole number increment that includes an amino acid position having avalue greater than 0.5 in the Hydrophilicity profile of FIG. 5; apeptide region of at least 5 amino acids of FIGS. 2 and 3 in any wholenumber increment that includes an amino acid position having a valueless than 0.5 in the Hydropathicity profile of FIG. 6; a peptide regionof at least 5 amino acids of FIGS. 2 and 3 in any whole number incrementthat includes an amino acid position having a value greater than 0.5 inthe Percent Accessible Residues profile of FIG. 7; a peptide region ofat least 5 amino acids of FIGS. 2 and 3 in any whole number incrementthat includes an amino acid position having a value greater than 0.5 inthe Average Flexibility profile on FIG. 8; and, a peptide region of atleast 5 amino acids of FIGS. 2 and 3 in any whole number increment thatincludes an amino acid position having a value greater than 0.5 in theBeta-turn profile of FIG. 9. Peptide immunogens of the invention canalso comprise nucleic acids that encode any of the forgoing.

All immunogens of the invention, peptide or nucleic acid, can beembodied in human unit dose form, or comprised by a composition thatincludes a pharmaceutical excipient compatible with human physiology.

The secondary structure of 24P4C12 variant 1, namely the predictedpresence and location of alpha helices, extended strands, and randomcoils, are predicted from the respective primary amino acid sequencesusing the HNN—Hierarchical Neural Network method (Guermeur, 1997,http://pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html), accessedfrom the ExPasy molecular biology server (http://www.expasy.ch/tools/).The analysis indicates that 24P4C12 variant 1 is composed of 53.94%alpha helix, 9.44% extended strand, and 36.62% random coil (FIG. 13 a).Analysis for the potential presence of transmembrane domains in 24P4C12variants were carried out using a variety of transmembrane predictionalgorithms accessed from the ExPasy molecular biology server(http://www.expasy.ch/tools/). Shown graphically are the results ofanalysis of variant 1 depicting the presence and location of 10transmembrane domains using the TMpred program (FIG. 13 b) and TMHMMprogram (FIG. 13 c). The results of each program, namely the amino acidsencoding the transmembrane domains are summarized in Table L.

Example 10 Generation of 24P4C12 Polyclonal Antibodies

Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Inaddition to immunizing with the full length 24P4C12 protein, computeralgorithms are employed in design of immunogens that, based on aminoacid sequence analysis contain characteristics of being antigenic andavailable for recognition by the immune system of the immunized host(see the Example entitled “Antigenicity Profiles”). Such regions wouldbe predicted to be hydrophilic, flexible, in beta-turn conformations,and be exposed on the surface of the protein (see, e.g., FIG. 5, FIG. 6,FIG. 7, FIG. 8, or FIG. 9 for amino acid profiles that indicate suchregions of 24P4C12 and variants).

For example, 24P4C12 recombinant bacterial fusion proteins or peptidescontaining hydrophilic, flexible, beta-turn regions of 24P4C12 variantproteins are used as antigens to generate polyclonal antibodies in NewZealand White rabbits. For example, such regions include, but are notlimited to, amino acids 1-34, amino acids 118-135, amino acids 194-224,amino acids 280-290, and amino acids 690-710, of 24P4C12 variants 1. Itis useful to conjugate the immunizing agent to a protein known to beimmunogenic in the mammal being immunized. Examples of such immunogenicproteins include, but are not limited to, keyhole limpet hemocyanin(KLH), serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. In one embodiment, a peptide encoding amino acids 1-14 of24P4C12 variant 1 was conjugated to KLH and used to immunize a rabbit.This antiserum exhibited a high titer to the peptide (>10,000) andrecognized 24P4C12 in transfected 293T cells by Western blot and flowcytometry (FIG. 24) and in stable recombinant PC3 cells by Western blotand immunohistochemistry (FIG. 25). Alternatively the immunizing agentmay include all or portions of the 24P4C12 variant proteins, analogs orfusion proteins thereof. For example, the 24P4C12 variant 1 amino acidsequence can be fused using recombinant DNA techniques to any one of avariety of fusion protein partners that are well known in the art, suchas glutathione-S-transferase (GST) and HIS tagged fusion proteins. Suchfusion proteins are purified from induced bacteria using the appropriateaffinity matrix.

In one embodiment, a GST-fusion protein encoding amino acids 379-453,encompassing the third predicted extracellular loop of variant 1, isproduced, purified, and used as immunogen. Other recombinant bacterialfusion proteins that may be employed include maltose binding protein,LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see thesection entitled “Production of 24P4C12 in Prokaryotic Systems” andCurrent Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M.Ausubul et al. eds., 1995; Linsley, P.S., Brady, W., Urnes, M.,Grosmaire, L., Damle, N., and Ledbetter, L. (1991) J. Exp. Med. 174,561-566).

In addition to bacterial derived fusion proteins, mammalian expressedprotein antigens are also used. These antigens are expressed frommammalian expression vectors such as the Tag5 and Fc-fusion vectors (seethe Example entitled “Production of Recombinant 24P4C12 in EukaryoticSystems”), and retains post-translational modifications such asglycosylations found in native protein. In two embodiments, thepredicted 1st and third extracellular loops of variant 1, amino acids59-227 and 379-453 respectively, were each cloned into the Tag5mammalian secretion vector and expressed in 293T cells (FIG. 26). Eachrecombinant protein is then purified by metal chelate chromatographyfrom tissue culture supernatants and/or lysates of 293T cells stablyexpressing the recombinant vector. The purified Tag5 24P4C12 protein isthen used as immunogen.

During the immunization protocol, it is useful to mix or emulsify theantigen in adjuvants that enhance the immune response of the hostanimal. Examples of adjuvants include, but are not limited to, completeFreund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate).

In a typical protocol, rabbits are initially immunized subcutaneouslywith up to 200 μg, typically 100-200 μg, of fusion protein or peptideconjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits arethen injected subcutaneously every two weeks with up to 200 μg,typically 100-200 μg, of the immunogen in incomplete Freund's adjuvant(IFA). Test bleeds are taken approximately 7-10 days following eachimmunization and used to monitor the titer of the antiserum by ELISA.

To test reactivity and specificity of immune serum, such as the rabbitserum derived from immunization with a KLH-conjugated peptide encodingamino acids 1-14 of variant 1, the full-length 24P4C12 variant 1 cDNA iscloned into pcDNA 3.1 myc-his or retroviral expression vectors(Invitrogen, see the Example entitled “Production of Recombinant 24P4C12in Eukaryotic Systems”). After transfection of the constructs into 293Tcells or transduction of PC3 with 24P4C12 retrovirus, cell lysates areprobed with the anti-24P4C12 serum and with anti-His antibody (SantaCruz Biotechnologies, Santa Cruz, Calif.) to determine specificreactivity to denatured 24P4C12 protein using the Western blottechnique. As shown in FIGS. 24 and 25 the antiserum specificallyrecognizes 24P4C12 protein in 293T and PC3 cells. In addition, theimmune serum is tested by fluorescence microscopy, flow cytometry, andimmunohistochemistry (FIG. 25) and immunoprecipitation against 293T andother recombinant 24P4C12-expressing cells to determine specificrecognition of native protein. Western blot, immunoprecipitation,fluorescent microscopy, immunohistochemistry and flow cytometrictechniques using cells that endogenously express 24P4C12 are alsocarried out to test reactivity and specificity.

Anti-serum from rabbits immunized with 24P4C12 variant fusion proteins,such as GST and MBP fusion proteins, are purified by depletion ofantibodies reactive to the fusion partner sequence by passage over anaffinity column containing the fusion partner either alone or in thecontext of an irrelevant fusion protein. For example, antiserum derivedfrom a GST-24P4C12 fusion protein encoding amino acids 379-453 ofvariant 1 is first purified by passage over a column of GST proteincovalently coupled to AffiGel matrix (BioRad, Hercules, Calif.). Theantiserum is then affinity purified by passage over a column composed ofa MBP-fusion protein also encoding amino acids 379-453 covalentlycoupled to Affigel matrix. The serum is then further purified by proteinG affinity chromatography to isolate the IgG fraction. Sera from otherHis-tagged antigens and peptide immunized rabbits as well as fusionpartner depleted sera are affinity purified by passage over a columnmatrix composed of the original protein immunogen or free peptide.

Example 11 Generation of 24P4C12 Monoclonal Antibodies (mAbs)

In one embodiment, therapeutic mAbs to 24P4C12 variants comprise thosethat react with epitopes specific for each variant protein or specificto sequences in common between the variants that would disrupt ormodulate the biological function of the 24P4C12 variants, for examplethose that would disrupt the interaction with ligands and substrates ordisrupt its biological activity. Immunogens for generation of such mAbsinclude those designed to encode or contain the entire 24P4C12 proteinvariant sequence, regions of the 24P4C12 protein variants predicted tobe antigenic from computer analysis of the amino acid sequence (see,e.g., FIG. 5, FIG. 6, FIG. 7, FIG. 8, or FIG. 9, and the Exampleentitled “Antigenicity Profiles”). Immunogens include peptides,recombinant bacterial proteins, and mammalian expressed Tag 5 proteinsand human and murine IgG FC fusion proteins. In addition, cellsengineered to express high levels of a respective 24P4C12 variant, suchas 293T-24P4C12 variant 1 or 300.19-24P4C12 variant 1 murine Pre-Bcells, are used to immunize mice.

To generate mAbs to a 24P4C12 variant, mice are first immunizedintraperitoneally (IP) with, typically, 10-50 μg of protein immunogen or10⁷ 24P4C12-expressing cells mixed in complete Freund's adjuvant. Miceare then subsequently immunized IP every 2-4 weeks with, typically,10-50 μg of protein immunogen or 10⁷ cells mixed in incomplete Freund'sadjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. Inone embodiment, mice were immunized as above with 300.19-24P4C12 cellsin complete and then incomplete Freund's adjuvant, and subsequentlysacrificed and the spleens harvested and used for fusion and hybridomageneration. As is can be seen in FIG. 27, 2 hybridomas were generatedwhose antibodies specifically recognize 24P4C12 protein expressed in293T cells by flow cytometry. In addition to the above protein andcell-based immunization strategies, a DNA-based immunization protocol isemployed in which a mammalian expression vector encoding a 24P4C12variant sequence is used to immunize mice by direct injection of theplasmid DNA. In one embodiment, a Tag5 mammalian secretion vectorencoding amino acids 59-227 of the variant 1 sequence (FIG. 26) was usedto immunize mice. Subsequent booster immunizations are then carried outwith the purified protein. In another example, the same amino acids arecloned into an Fc-fusion secretion vector in which the 24P4C12 variant 1sequence is fused at the amino-terminus to an IgK leader sequence and atthe carboxyl-terminus to the coding sequence of the human or murine IgGFc region. This recombinant vector is then used as immunogen. Theplasmid immunization protocols are used in combination with purifiedproteins as above and with cells expressing the respective 24P4C12variant.

During the immunization protocol, test bleeds are taken 7-10 daysfollowing an injection to monitor titer and specificity of the immuneresponse. Once appropriate reactivity and specificity is obtained asdetermined by ELISA, Western blotting, immunoprecipitation, fluorescencemicroscopy, immunohistochemistry, and flow cytometric analyses, fusionand hybridoma generation is then carried out with established procedureswell known in the art (see, e.g., Harlow and Lane, 1988).

In one embodiment for generating 24P4C12 variant 8 specific monoclonalantibodies, a peptide encoding amino acids 643-654 (RNPITPTGHVFQ) (SEQID NO: 46) of 24P4C12 variant 8 is synthesized, coupled to KLH and usedas immunogen. Balb C mice are initially immunized intraperitoneally with25 μg of the KLH-24P4C12 variant 8 peptide mixed in complete Freund'sadjuvant. Mice are subsequently immunized every two weeks with 25 μg ofthe antigen mixed in incomplete Freund's adjuvant for a total of threeimmunizations. ELISA using the free peptide determines the reactivity ofserum from immunized mice. Reactivity and specificity of serum to fulllength 24P4C12 variant 8 protein is monitored by Western blotting,immunoprecipitation and flow cytometry using 293T cells transfected withan expression vector encoding the 24P4C12 variant 8 cDNA compared tocells transfected with the other 24P4C12 variants (see e.g., the Exampleentitled “Production of Recombinant 24P4C12 in Eukaryotic Systems”).Other recombinant 24P4C12 variant 8-expressing cells or cellsendogenously expressing 24P4C12 variant 8 are also used. Mice showingthe strongest specific reactivity to 24P4C12 variant 8 are rested andgiven a final injection of antigen in PBS and then sacrificed four dayslater. The spleens of the sacrificed mice are harvested and fused toSPO/2 myeloma cells using standard procedures (Harlow and Lane, 1988).Supernatants from HAT selected growth wells are screened by ELISA,Western blot, immunoprecipitation, fluorescent microscopy, and flowcytometry to identify 24P4C12 variant 8-specific antibody-producingclones. A similar strategy is also used to derive 24P4C12 variant9-specific antibodies using a peptide encompassing amino acids 379-388(PLPTOPATLG) (SEQ ID NO: 47).

The binding affinity of a 24P4C12 monoclonal antibody is determinedusing standard technologies. Affinity measurements quantify the strengthof antibody to epitope binding and are used to help define which 24P4C12monoclonal antibodies preferred for diagnostic or therapeutic use, asappreciated by one of skill in the art. The BIAcore system (Uppsala,Sweden) is a preferred method for determining binding affinity. TheBIAcore system uses surface plasmon resonance (SPR, Welford K. 1991,Opt. Quant. Elect. 23:1; Morton and Myszka, 1998, Methods in Enzymology295: 268) to monitor bimolecular interactions in real time. BIAcoreanalysis conveniently generates association rate constants, dissociationrate constants, equilibrium dissociation constants, and affinityconstants.

Example 12 HLA Class I and Class II Binding Assays

HLA class I and class II binding assays using purified HLA molecules areperformed in accordance with disclosed protocols (e.g., PCT publicationsWO 94/20127 and WO 94/03205; Sidney et al., Current Protocols inImmunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995);Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, purified MHCmolecules (5 to 500 nM) are incubated with various unlabeled peptideinhibitors and 1-10 nM Q ¹²⁵I-radiolabeled probe peptides as described.Following incubation, MHC-peptide complexes are separated from freepeptide by gel filtration and the fraction of peptide bound isdetermined. Typically, in preliminary experiments, each MHC preparationis titered in the presence of fixed amounts of radiolabeled peptides todetermine the concentration of HLA molecules necessary to bind 10-20% ofthe total radioactivity. All subsequent inhibition and direct bindingassays are performed using these HLA concentrations.

Since under these conditions [label]<[HLA] and IC₅₀≧[HLA], the measuredIC₅₀ values are reasonable approximations of the true K_(D) values.Peptide inhibitors are typically tested at concentrations ranging from120 μg/ml to 1.2 ng/ml, and are tested in two to four completelyindependent experiments. To allow comparison of the data obtained indifferent experiments, a relative binding figure is calculated for eachpeptide by dividing the IC₅₀ of a positive control for inhibition by theIC₅₀ for each tested peptide (typically unlabeled versions of theradiolabeled probe peptide). For database purposes, and inter-experimentcomparisons, relative binding values are compiled. These values cansubsequently be converted back into IC₅₀ nM values by dividing the IC₅₀nM of the positive controls for inhibition by the relative binding ofthe peptide of interest. This method of data compilation is accurate andconsistent for comparing peptides that have been tested on differentdays, or with different lots of purified MHC.

Binding assays as outlined above may be used to analyze HLA supermotifand/or HLA motif-bearing peptides (see Table IV).

Example 13 Identification of HLA Supermotif- and Motif-Bearing CTLCandidate Epitopes

HLA vaccine compositions of the invention can include multiple epitopes.The multiple epitopes can comprise multiple HLA supermotifs or motifs toachieve broad population coverage. This example illustrates theidentification and confirmation of supermotif- and motif-bearingepitopes for the inclusion in such a vaccine composition. Calculation ofpopulation coverage is performed using the strategy described below.

Computer Searches and Algorithms for Identification of Supermotif and/orMotif-Bearing Epitopes

The searches performed to identify the motif-bearing peptide sequencesin the Example entitled “Antigenicity Profiles” and Tables VIII-XXI andXXII-XLIX employ the protein sequence data from the gene product of24P4C12 set forth in FIGS. 2 and 3, the specific search peptides used togenerate the tables are listed in Table VII.

Computer searches for epitopes bearing HLA Class I or Class IIsupermotifs or motifs are performed as follows. All translated 24P4C12protein sequences are analyzed using a text string search softwareprogram to identify potential peptide sequences containing appropriateHLA binding motifs; such programs are readily produced in accordancewith information in the art in view of known motif/supermotifdisclosures. Furthermore, such calculations can be made mentally.

Identified A2-, A3-, and DR-supermotif sequences are scored usingpolynomial algorithms to predict their capacity to bind to specificHLA-Class I or Class II molecules. These polynomial algorithms accountfor the impact of different amino acids at different positions, and areessentially based on the premise that the overall affinity (or AG) ofpeptide-HLA molecule interactions can be approximated as a linearpolynomial function of the type:

“ΔG”=a _(1i) ×a _(2i) ×a _(3i) . . . ×a _(ni)

where a_(ji) is a coefficient which represents the effect of thepresence of a given amino acid (j) at a given position (i) along thesequence of a peptide of n amino acids. The crucial assumption of thismethod is that the effects at each position are essentially independentof each other (i.e., independent binding of individual side-chains).When residue j occurs at position i in the peptide, it is assumed tocontribute a constant amount i to the free energy of binding of thepeptide irrespective of the sequence of the rest of the peptide.

The method of derivation of specific algorithm coefficients has beendescribed in Gulukota et al., J. Mol. Biol. 267:1258-126, 1997; (seealso Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood et al.,J. Immunol. 160:3363-3373, 1998). Briefly, for all i positions, anchorand non-anchor alike, the geometric mean of the average relative binding(ARB) of all peptides carrying j is calculated relative to the remainderof the group, and used as the estimate of j_(i). For Class II peptides,if multiple alignments are possible, only the highest scoring alignmentis utilized, following an iterative procedure. To calculate an algorithmscore of a given peptide in a test set, the ARB values corresponding tothe sequence of the peptide are multiplied. If this product exceeds achosen threshold, the peptide is predicted to bind. Appropriatethresholds are chosen as a function of the degree of stringency ofprediction desired.

Selection of HLA-A2 Supertype Cross-Reactive Peptides

Protein sequences from 24P4C12 are scanned utilizing motifidentification software, to identify 8-, 9- 10- and 11-mer sequencescontaining the HLA-A2-supermotif main anchor specificity. Typically,these sequences are then scored using the protocol described above andthe peptides corresponding to the positive-scoring sequences aresynthesized and tested for their capacity to bind purified HLA-A*0201molecules in vitro (HLA-A*0201 is considered a prototype A2 supertypemolecule).

These peptides are then tested for the capacity to bind to additionalA2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). Peptidesthat bind to at least three of the five A2-supertype alleles tested aretypically deemed A2-supertype cross-reactive binders. Preferred peptidesbind at an affinity equal to or less than 500 nM to three or more HLA-A2supertype molecules.

Selection of HLA-A3 Supermotif-Bearing Epitopes

The 24P4C12 protein sequence(s) scanned above is also examined for thepresence of peptides with the HLA-A3-supermotif primary anchors.Peptides corresponding to the HLA A3 supermotif-bearing sequences arethen synthesized and tested for binding to HLA-A*0301 and HLA-A*L 1101molecules, the molecules encoded by the two most prevalent A3-supertypealleles. The peptides that bind at least one of the two alleles withbinding affinities of ≦500 nM, often ≦200 nM, are then tested forbinding cross-reactivity to the other common A3-supertype alleles (e.g.,A*3101, A*3301, and A*6801) to identify those that can bind at leastthree of the five HLA-A3-supertype molecules tested.

Selection of HLA-B7 Supermotif Bearing Epitopes

The 24P4C12 protein(s) scanned above is also analyzed for the presenceof 8-, 9- 10-, or 11-mer peptides with the HLA-B7-supermotif.Corresponding peptides are synthesized and tested for binding toHLA-B*0702, the molecule encoded by the most common B7-supertype allele(i.e., the prototype B7 supertype allele). Peptides binding B*0702 withIC₅₀ of ≦500 nM are identified using standard methods. These peptidesare then tested for binding to other common B7-supertype molecules(e.g., B*3501, B*5101, B*5301, and B*5401). Peptides capable of bindingto three or more of the five B7-supertype alleles tested are therebyidentified.

Selection of A1 and A24 Motif-Bearing Epitopes

To further increase population coverage, HLA-A1 and -A24 epitopes canalso be incorporated into vaccine compositions. An analysis of the24P4C12 protein can also be performed to identify HLA-A1- andA24-motif-containing sequences.

High affinity and/or cross-reactive binding epitopes that bear othermotif and/or supermotifs are identified using analogous methodology.

Example 14 Confirmation of Immunogenicity

Cross-reactive candidate CTL A2-supermotif-bearing peptides that areidentified as described herein are selected to confirm in vitroimmunogenicity. Confirmation is performed using the followingmethodology:

Target Cell Lines for Cellular Screening:

The 0.221A2.1 cell line, produced by transferring the HLA-A2.1 gene intothe HLA-A, -B, -C null mutant human B-lymphoblastoid cell line 721.221,is used as the peptide-loaded target to measure activity ofHLA-A2.1-restricted CTL. This cell line is grown in RPMI-1640 mediumsupplemented with antibiotics, sodium pyruvate, nonessential amino acidsand 10% (v/v) heat inactivated FCS. Cells that express an antigen ofinterest, or transfectants comprising the gene encoding the antigen ofinterest, can be used as target cells to confirm the ability ofpeptide-specific CTLs to recognize endogenous antigen.

Primary CTL Induction Cultures:

Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI with 30μg/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640plus 5% AB human serum, non-essential amino acids, sodium pyruvate,L-glutamine and penicillin/streptomycin). The monocytes are purified byplating 10×10⁶ PBMC/well in a 6-well plate. After 2 hours at 37° C., thenon-adherent cells are removed by gently shaking the plates andaspirating the supernatants. The wells are washed a total of three timeswith 3 ml RPMI to remove most of the non-adherent and loosely adherentcells. Three ml of complete medium containing 50 ng/ml of GM-CSF and1,000 U/ml of IL-4 are then added to each well. TNFα is added to the DCson day 6 at 75 ng/ml and the cells are used for CTL induction cultureson day 7.

Induction of CTL with DC and Peptide: CD8+ T-cells are isolated bypositive selection with Dynal immunomagnetic beads (Dynabeads® M-450)and the Detacha-Bead® reagent. Typically about 200-250×10⁶ PBMC areprocessed to obtain 24×10⁶ CD8+ T-cells (enough for a 48-well plateculture). Briefly, the PBMCs are thawed in RPMI with 30 μg/ml DNAse,washed once with PBS containing 1% human AB serum and resuspended inPBS/1% AB serum at a concentration of 20×10⁶ cells/ml. The magneticbeads are washed 3 times with PBS/AB serum, added to the cells (140μlbeads/20×10⁶ cells) and incubated for 1 hour at 4° C. with continuousmixing. The beads and cells are washed 4× with PBS/AB serum to removethe nonadherent cells and resuspended at 100×10⁶ cells/ml (based on theoriginal cell number) in PBS/AB serum containing 100 μl/ml Detacha-Bead®reagent and 30 μg/ml DNAse. The mixture is incubated for 1 hour at roomtemperature with continuous mixing. The beads are washed again withPBS/AB/DNAse to collect the CD8+ T-cells. The DC are collected andcentrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1%BSA, counted and pulsed with 40 μg/ml of peptide at a cell concentrationof 1-2×10⁶/ml in the presence of 3 μg/ml β₂-microglobulin for 4 hours at20° C. The DC are then irradiated (4,200 rads), washed 1 time withmedium and counted again.

Setting up induction cultures: 0.25 ml cytokine-generated DC (at 1×10⁵cells/ml) are co-cultured with 0.25 ml of CD8+ T-cells (at 2×10⁶cell/ml) in each well of a 48-well plate in the presence of 10 ng/ml ofIL-7. Recombinant human IL-10 is added the next day at a finalconcentration of 10 ng/ml and rhuman IL-2 is added 48 hours later at 10IU/ml.

Restimulation of the induction cultures with peptide-pulsed adherentcells: Seven and fourteen days after the primary induction, the cellsare restimulated with peptide-pulsed adherent cells. The PBMCs arethawed and washed twice with RPMI and DNAse. The cells are resuspendedat 5×10⁶ cells/ml and irradiated at ˜4200 rads. The PBMCs are plated at2×10⁶ in 0.5 ml complete medium per well and incubated for 2 hours at37° C. The plates are washed twice with RPMI by tapping the plate gentlyto remove the nonadherent cells and the adherent cells pulsed with 1μg/ml of peptide in the presence of 3 μg/ml B2 microglobulin in 0.25 mlRPMI/5% AB per well for 2 hours at 37° C. Peptide solution from eachwell is aspirated and the wells are washed once with RPMI. Most of themedia is aspirated from the induction cultures (CD8+ cells) and broughtto 0.5 ml with fresh media. The cells are then transferred to the wellscontaining the peptide-pulsed adherent cells. Twenty four hours laterrecombinant human IL-10 is added at a final concentration of 10 ng/mland recombinant human IL2 is added the next day and again 2-3 days laterat 50 IU/ml (Tsai et al., Critical Reviews in Immunology 18 (1-2):65-75,1998). Seven days later, the cultures are assayed for CTL activity in a⁵¹Cr release assay. In some experiments the cultures are assayed forpeptide-specific recognition in the in situ IFNγ ELISA at the time ofthe second restimulation followed by assay of endogenous recognition 7days later. After expansion, activity is measured in both assays for aside-by-side comparison.

Measurement of CTL Lytic Activity by ⁵¹Cr Release.

Seven days after the second restimulation, cytotoxicity is determined ina standard (5 hr) ⁵¹Cr release assay by assaying individual wells at asingle E:T. Peptide-pulsed targets are prepared by incubating the cellswith lop g/ml peptide overnight at 37° C.

Adherent target cells are removed from culture flasks with trypsin-EDTA.Target cells are labeled with 200 μCi of ⁵¹Cr sodium chromate (Dupont,Wilmington, Del.) for 1 hour at 37° C. Labeled target cells areresuspended at 10⁶ per ml and diluted 1:10 with K562 cells at aconcentration of 3.3×10⁶/ml (an NK-sensitive erythroblastoma cell lineused to reduce non-specific lysis). Target cells (100 μl) and effectors(100 μl) are plated in 96 well round-bottom plates and incubated for 5hours at 37° C. At that time, 100 μl of supernatant are collected fromeach well and percent lysis is determined according to the formula:

[(cpm of the test sample−cpm of the spontaneous ⁵¹Cr releasesample)/(cpm of the maximal ⁵¹Cr release sample−cpm of the spontaneous⁵¹Cr release sample)]×100.

Maximum and spontaneous release are determined by incubating the labeledtargets with 1% Triton X-100 and media alone, respectively. A positiveculture is defined as one in which the specific lysis(sample-background) is 10% or higher in the case of individual wells andis 15% or more at the two highest E:T ratios when expanded cultures areassayed.

In situ Measurement of Human IFNγ Production as an Indicator ofPeptide-Specific and Endogenous Recognition

Immulon 2 plates are coated with mouse anti-human IFNγ monoclonalantibody (4 μg/ml 0.1M NaHCO₃, pH8.2) overnight at 4° C. The plates arewashed with Ca²⁺, Mg²⁺-free PBS/0.05% Tween 20 and blocked with PBS/10%FCS for two hours, after which the CTLs (100 μl/well) and targets (100μl/well) are added to each well, leaving empty wells for the standardsand blanks (which received media only). The target cells, eitherpeptide-pulsed or endogenous targets, are used at a concentration of1×10⁶ cells/ml. The plates are incubated for 48 hours at 37° C. with 5%CO₂.

Recombinant human IFN-gamma is added to the standard wells starting at400 pg or 1200 pg/100 microliter/well and the plate incubated for twohours at 37° C. The plates are washed and 100 μl of biotinylated mouseanti-human IFN-gamma monoclonal antibody (2 microgram/ml in PBS/3%FCS/0.05% Tween 20) are added and incubated for 2 hours at roomtemperature. After washing again, 100 microliter HRP-streptavidin(1:4000) are added and the plates incubated for one hour at roomtemperature. The plates are then washed 6× with wash buffer, 100microliter/well developing solution (TMB 1:1) are added, and the platesallowed to develop for 5-15 minutes. The reaction is stopped with 50microliter/well 1 M H₃PO₄ and read at OD450. A culture is consideredpositive if it measured at least 50 pg of IFN-gamma/well abovebackground and is twice the background level of expression.

CTL Expansion.

Those cultures that demonstrate specific lytic activity againstpeptide-pulsed targets and/or tumor targets are expanded over a two weekperiod with anti-CD3. Briefly, 5×10⁴ CD8+ cells are added to a T25 flaskcontaining the following: 1×10⁶ irradiated (4,200 rad) PBMC (autologousor allogeneic) per ml, 2×10⁵ irradiated (8,000 rad) EBV-transformedcells per ml, and OKT3 (anti-CD3) at 30 ng per ml in RPMI-1640containing 10% (v/v) human AB serum, non-essential amino acids, sodiumpyruvate, 25 μM 2-mercaptoethanol, L-glutamine andpenicillin/streptomycin. Recombinant human IL2 is added 24 hours laterat a final concentration of 200 IU/ml and every three days thereafterwith fresh media at 50 IU/ml. The cells are split if the cellconcentration exceeds 1×10⁶/ml and the cultures are assayed between days13 and 15 at E:T ratios of 30, 10, 3 and 1.1 in the ⁵¹Cr release assayor at 1×10⁶/ml in the in situ IFNγ assay using the same targets asbefore the expansion.

Cultures are expanded in the absence of anti-CD3⁺ as follows. Thosecultures that demonstrate specific lytic activity against peptide andendogenous targets are selected and 5×10⁴ CD8⁺ cells are added to a T25flask containing the following: 1×10⁶ autologous PBMC per ml which havebeen peptide-pulsed with 10 μg/ml peptide for two hours at 37° C. andirradiated (4,200 rad); 2×10⁵ irradiated (8,000 rad) EBV-transformedcells per ml RPMI-1640 containing 10% (v/v) human AB serum,non-essential AA, sodium pyruvate, 25 mM 2-ME, L-glutamine andgentamicin.

Immunogenicity of A2 Supermotif-Bearing Peptides

A2-supermotif cross-reactive binding peptides are tested in the cellularassay for the ability to induce peptide-specific CTL in normalindividuals. In this analysis, a peptide is typically considered to bean epitope if it induces peptide-specific CTLs in at least individuals,and preferably, also recognizes the endogenously expressed peptide.

Immunogenicity can also be confirmed using PBMCs isolated from patientsbearing a tumor that expresses 24P4C12. Briefly, PBMCs are isolated frompatients, re-stimulated with peptide-pulsed monocytes and assayed forthe ability to recognize peptide-pulsed target cells as well astransfected cells endogenously expressing the antigen.

Evaluation of A*03/A11 Immunogenicity

HLA-A3 supermotif-bearing cross-reactive binding peptides are alsoevaluated for immunogenicity using methodology analogous for that usedto evaluate the immunogenicity of the HLA-A2 supermotif peptides.

Evaluation of B7 Immunogenicity

Immunogenicity screening of the B7-supertype cross-reactive bindingpeptides identified as set forth herein are confirmed in a manneranalogous to the confirmation of A2- and A3-supermotif-bearing peptides.

Peptides bearing other supermotifs/motifs, e.g., HLA-A1, HLA-A24 etc.are also confirmed using similar methodology

Example 15 Implementation of the Extended Supermotif to Improve theBinding Capacity of Native Epitopes by Creating Analogs

HLA motifs and supermotifs (comprising primary and/or secondaryresidues) are useful in the identification and preparation of highlycross-reactive native peptides, as demonstrated herein. Moreover, thedefinition of HLA motifs and supermotifs also allows one to engineerhighly cross-reactive epitopes by identifying residues within a nativepeptide sequence which can be analoged to confer upon the peptidecertain characteristics, e.g. greater cross-reactivity within the groupof HLA molecules that comprise a supertype, and/or greater bindingaffinity for some or all of those HLA molecules. Examples of analogingpeptides to exhibit modulated binding affinity are set forth in thisexample.

Analoging at Primary Anchor Residues

Peptide engineering strategies are implemented to further increase thecross-reactivity of the epitopes. For example, the main anchors ofA2-supermotif-bearing peptides are altered, for example, to introduce apreferred L, I, V, or M at position 2, and I or V at the C-terminus.

To analyze the cross-reactivity of the analog peptides, each engineeredanalog is initially tested for binding to the prototype A2 supertypeallele A*0201, then, if A*0201 binding capacity is maintained, forA2-supertype cross-reactivity.

Alternatively, a peptide is confirmed as binding one or all supertypemembers and then analoged to modulate binding affinity to any one (ormore) of the supertype members to add population coverage.

The selection of analogs for immunogenicity in a cellular screeninganalysis is typically further restricted by the capacity of the parentwild type (WT) peptide to bind at least weakly, i.e., bind at an IC₅₀ of500 nM or less, to three of more A2 supertype alleles. The rationale forthis requirement is that the WT peptides must be present endogenously insufficient quantity to be biologically relevant. Analoged peptides havebeen shown to have increased immunogenicity and cross-reactivity by Tcells specific for the parent epitope (see, e.g., Parkhurst et al., J.Immunol. 157:2539, 1996; and Pogue et al., Proc. Natl. Acad. Sci. USA92:8166, 1995).

In the cellular screening of these peptide analogs, it is important toconfirm that analog-specific CTLs are also able to recognize thewild-type peptide and, when possible, target cells that endogenouslyexpress the epitope.

Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides

Analogs of HLA-A3 supermotif-bearing epitopes are generated usingstrategies similar to those employed in analoging HLA-A2supermotif-bearing peptides. For example, peptides binding to 3/5 of theA3-supertype molecules are engineered at primary anchor residues topossess a preferred residue (V, S, M, or A) at position 2.

The analog peptides are then tested for the ability to bind A*03 andA*11 (prototype A3 supertype alleles). Those peptides that demonstrate≦500 nM binding capacity are then confirmed as having A3-supertypecross-reactivity.

Similarly to the A2- and A3-motif bearing peptides, peptides binding 3or more B7-supertype alleles can be improved, where possible, to achieveincreased cross-reactive binding or greater binding affinity or bindinghalf life. B7 supermotif-bearing peptides are, for example, engineeredto possess a preferred residue (V, I, L, or F) at the C-terminal primaryanchor position, as demonstrated by Sidney et al. (J. Immunol.157:3480-3490, 1996).

Analoging at primary anchor residues of other motif and/orsupermotif-bearing epitopes is performed in a like manner.

The analog peptides are then be confirmed for immunogenicity, typicallyin a cellular screening assay. Again, it is generally important todemonstrate that analog-specific CTLs are also able to recognize thewild-type peptide and, when possible, targets that endogenously expressthe epitope.

Analoging at Secondary Anchor Residues

Moreover, HLA supermotifs are of value in engineering highlycross-reactive peptides and/or peptides that bind HLA molecules withincreased affinity by identifying particular residues at secondaryanchor positions that are associated with such properties. For example,the binding capacity of a B7 supermotif-bearing peptide with an Fresidue at position 1 is analyzed. The peptide is then analoged to, forexample, substitute L for F at position 1. The analoged peptide isevaluated for increased binding affinity, binding half life and/orincreased cross-reactivity. Such a procedure identifies analogedpeptides with enhanced properties.

Engineered analogs with sufficiently improved binding capacity orcross-reactivity can also be tested for immunogenicity inHLA-B7-transgenic mice, following for example, IFA immunization orlipopeptide immunization. Analoged peptides are additionally tested forthe ability to stimulate a recall response using PBMC from patients with24P4C12-expressing tumors.

Other Analoging Strategies

Another form of peptide analoging, unrelated to anchor positions,involves the substitution of a cysteine with α-amino butyric acid. Dueto its chemical nature, cysteine has the propensity to form disulfidebridges and sufficiently alter the peptide structurally so as to reducebinding capacity. Substitution of α-amino butyric acid for cysteine notonly alleviates this problem, but has been shown to improve binding andcrossbinding capabilities in some instances (see, e.g., the review bySette et al., In: Persistent Viral Infections, Eds. R. Ahmed and 1.Chen, John Wiley & Sons, England, 1999).

Thus, by the use of single amino acid substitutions, the bindingproperties and/or cross-reactivity of peptide ligands for HLA supertypemolecules can be modulated.

Example 16 Identification and Confirmation of 24P4C12-Derived Sequenceswith HLA-DR Binding Motifs

Peptide epitopes bearing an HLA class II supermotif or motif areidentified and confirmed as outlined below using methodology similar tothat described for HLA Class I peptides.

Selection of HLA-DR-Supermotif-Bearing Epitopes.

To identify 24P4C12-derived, HLA class II HTL epitopes, a 24P4C12antigen is analyzed for the presence of sequences bearing anHLA-DR-motif or supermotif. Specifically, 15-mer sequences are selectedcomprising a DR-supermotif, comprising a 9-mer core, and three-residueN- and C-terminal flanking regions (15 amino acids total).

Protocols for predicting peptide binding to DR molecules have beendeveloped (Southwood et al., J. Immunol. 160:3363-3373, 1998). Theseprotocols, specific for individual DR molecules, allow the scoring, andranking, of 9-mer core regions. Each protocol not only scores peptidesequences for the presence of DR-supermotif primary anchors (i.e., atposition 1 and position 6) within a 9-mer core, but additionallyevaluates sequences for the presence of secondary anchors. Usingallele-specific selection tables (see, e.g., Southwood et al., ibid.),it has been found that these protocols efficiently select peptidesequences with a high probability of binding a particular DR molecule.Additionally, it has been found that performing these protocols intandem, specifically those for DR1, DR4w4, and DR7, can efficientlyselect DR cross-reactive peptides.

The 24P4C12-derived peptides identified above are tested for theirbinding capacity for various common HLA-DR molecules. All peptides areinitially tested for binding to the DR molecules in the primary panel:DR1, DR4w4, and DR7. Peptides binding at least two of these three DRmolecules are then tested for binding to DR2w2 β1, DR2w2β2, DR6w19, andDR9 molecules in secondary assays. Finally, peptides binding at leasttwo of the four secondary panel DR molecules, and thus cumulatively atleast four of seven different DR molecules, are screened for binding toDR4w15, DR5w11, and DR8w2 molecules in tertiary assays. Peptides bindingat least seven of the ten DR molecules comprising the primary,secondary, and tertiary screening assays are considered cross-reactiveDR binders. 24P4C12-derived peptides found to bind common HLA-DR allelesare of particular interest.

Selection of DR3 Motif Peptides

Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, andHispanic populations, DR3 binding capacity is a relevant criterion inthe selection of HTL epitopes. Thus, peptides shown to be candidates mayalso be assayed for their DR3 binding capacity. However, in view of thebinding specificity of the DR3 motif, peptides binding only to DR3 canalso be considered as candidates for inclusion in a vaccine formulation.

To efficiently identify peptides that bind DR3, target 24P4C12 antigensare analyzed for sequences carrying one of the two DR3-specific bindingmotifs reported by Geluk et al. (J. Immunol. 152:5742-5748, 1994). Thecorresponding peptides are then synthesized and confirmed as having theability to bind DR3 with an affinity of 1 μM or better, i.e., less than1 μM. Peptides are found that meet this binding criterion and qualify asHLA class II high affinity binders.

DR3 binding epitopes identified in this manner are included in vaccinecompositions with DR supermotif-bearing peptide epitopes.

Similarly to the case of HLA class I motif-bearing peptides, the classII motif-bearing peptides are analoged to improve affinity orcross-reactivity. For example, aspartic acid at position 4 of the 9-mercore sequence is an optimal residue for DR3 binding, and substitutionfor that residue often improves DR 3 binding.

Example 17 Immunogenicity of 24P4C12-Derived HTL Epitopes

This example determines immunogenic DR supermotif- and DR3 motif-bearingepitopes among those identified using the methodology set forth herein.

Immunogenicity of HTL epitopes are confirmed in a manner analogous tothe determination of immunogenicity of CTL epitopes, by assessing theability to stimulate HTL responses and/or by using appropriatetransgenic mouse models. Immunogenicity is determined by screening for:1.) in vitro primary induction using normal PBMC or 2.) recall responsesfrom patients who have 24P4C12-expressing tumors.

Example 18 Calculation of Phenotypic Frequencies of HLA-Supertypes inVarious Ethnic Backgrounds to Determine Breadth of Population Coverage

This example illustrates the assessment of the breadth of populationcoverage of a vaccine composition comprised of multiple epitopescomprising multiple supermotifs and/or motifs.

In order to analyze population coverage, gene frequencies of HLA allelesare determined. Gene frequencies for each HLA allele are calculated fromantigen or allele frequencies utilizing the binomial distributionformulae gf=1−(SQRT(1−af)) (see, e.g., Sidney et al., Human Immunol.45:79-93, 1996). To obtain overall phenotypic frequencies, cumulativegene frequencies are calculated, and the cumulative antigen frequenciesderived by the use of the inverse formula [af=1−(1−Cgf)²].

Where frequency data is not available at the level of DNA typing,correspondence to the serologically defined antigen frequencies isassumed. To obtain total potential supertype population coverage nolinkage disequilibrium is assumed, and only alleles confirmed to belongto each of the supertypes are included (minimal estimates). Estimates oftotal potential coverage achieved by inter-loci combinations are made byadding to the A coverage the proportion of the non-A covered populationthat could be expected to be covered by the B alleles considered (e.g.,total=A+B*(1−A)). Confirmed members of the A3-like supertype are A3,A11, A31, A*3301, and A*6801. Although the A3-like supertype may alsoinclude A34, A66, and A*7401, these alleles were not included in overallfrequency calculations. Likewise, confirmed members of the A2-likesupertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206,A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmedalleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601,B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, andB*5602).

Population coverage achieved by combining the A2-, A3- and B7-supertypesis approximately 86% in five major ethnic groups. Coverage may beextended by including peptides bearing the A1 and A24 motifs. Onaverage, A1 is present in 12% and A24 in 29% of the population acrossfive different major ethnic groups (Caucasian, North American Black,Chinese, Japanese, and Hispanic). Together, these alleles arerepresented with an average frequency of 39% in these same ethnicpopulations. The total coverage across the major ethnicities when A1 andA24 are combined with the coverage of the A2-, A3- and B7-supertypealleles is >95%, see, e.g., Table IV (G). An analogous approach can beused to estimate population coverage achieved with combinations of classII motif-bearing epitopes.

Immunogenicity studies in humans (e.g., Bertoni et al., J. Clin. Invest.100:503, 1997; Doolan et al., Immunity 7:97, 1997; and Threlkeld et al.,J. Immunol. 159:1648, 1997) have shown that highly cross-reactivebinding peptides are almost always recognized as epitopes. The use ofhighly cross-reactive binding peptides is an important selectioncriterion in identifying candidate epitopes for inclusion in a vaccinethat is immunogenic in a diverse population.

With a sufficient number of epitopes (as disclosed herein and from theart), an average population coverage is predicted to be greater than 95%in each of five major ethnic populations. The game theory Monte Carlosimulation analysis, which is known in the art (see e.g., Osborne, M. J.and Rubinstein, A. “A course in game theory” MIT Press, 1994), can beused to estimate what percentage of the individuals in a populationcomprised of the Caucasian, North American Black, Japanese, Chinese, andHispanic ethnic groups would recognize the vaccine epitopes describedherein. A preferred percentage is 90%. A more preferred percentage is95%.

Example 19 CTL Recognition of Endogenously Processed Antigens AfterPriming

This example confirms that CTL induced by native or analoged peptideepitopes identified and selected as described herein recognizeendogenously synthesized, i.e., native antigens.

Effector cells isolated from transgenic mice that are immunized withpeptide epitopes, for example HLA-A2 supermotif-bearing epitopes, arere-stimulated in vitro using peptide-coated stimulator cells. Six dayslater, effector cells are assayed for cytotoxicity and the cell linesthat contain peptide-specific cytotoxic activity are furtherre-stimulated. An additional six days later, these cell lines are testedfor cytotoxic activity on ⁵¹Cr labeled Jurkat-A2.1/K^(b) target cells inthe absence or presence of peptide, and also tested on ⁵¹Cr labeledtarget cells bearing the endogenously synthesized antigen, i.e. cellsthat are stably transfected with 24P4C12 expression vectors.

The results demonstrate that CTL lines obtained from animals primed withpeptide epitope recognize endogenously synthesized 24P4C12 antigen. Thechoice of transgenic mouse model to be used for such an analysis dependsupon the epitope(s) that are being evaluated. In addition toHLA-A*0201/K^(b) transgenic mice, several other transgenic mouse modelsincluding mice with human A11, which may also be used to evaluate A3epitopes, and B7 alleles have been characterized and others (e.g.,transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 andHLA-DR3 mouse models have also been developed, which may be used toevaluate HTL epitopes.

Example 20 Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice

This example illustrates the induction of CTLs and HTLs in transgenicmice, by use of a 24P4C12-derived CTL and HTL peptide vaccinecompositions. The vaccine composition used herein comprise peptides tobe administered to a patient with a 24P4C12-expressing tumor. Thepeptide composition can comprise multiple CTL and/or HTL epitopes. Theepitopes are identified using methodology as described herein. Thisexample also illustrates that enhanced immunogenicity can be achieved byinclusion of one or more HTL epitopes in a CTL vaccine composition; sucha peptide composition can comprise an HTL epitope conjugated to a CTLepitope. The CTL epitope can be one that binds to multiple HLA familymembers at an affinity of 500 nM or less, or analogs of that epitope.The peptides may be lipidated, if desired.

Immunization procedures: Immunization of transgenic mice is performed asdescribed (Alexander et al., J. Immunol. 159:4753-4761, 1997). Forexample, A2/K^(b) mice, which are transgenic for the human HLA A2.1allele and are used to confirm the immunogenicity of HLA-A*0201 motif-or HLA-A2 supermotif-bearing epitopes, and are primed subcutaneously(base of the tail) with a 0.1 ml of peptide in Incomplete Freund'sAdjuvant, or if the peptide composition is a lipidated CTL/HTLconjugate, in DMSO/saline, or if the peptide composition is apolypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days afterpriming, splenocytes obtained from these animals are restimulated withsyngenic irradiated LPS-activated lymphoblasts coated with peptide.

Cell lines: Target cells for peptide-specific cytotoxicity assays areJurkat cells transfected with the HLA-A2.1/K^(b) chimeric gene (e.g.,Vitiello et al., J. Exp. Med. 173:1007, 1991)

In vitro CTL activation: One week after priming, spleen cells (30×10⁶cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000rads), peptide coated lymphoblasts (10×10⁶ cells/flask) in 10 ml ofculture medium/T25 flask. After six days, effector cells are harvestedand assayed for cytotoxic activity.

Assay for cytotoxic activity: Target cells (1.0 to 1.5×10⁶) areincubated at 37° C. in the presence of 200 μl of ⁵¹Cr. After 60 minutes,cells are washed three times and resuspended in R10 medium. Peptide isadded where required at a concentration of 1 μg/ml. For the assay, 10⁴⁵¹Cr-labeled target cells are added to different concentrations ofeffector cells (final volume of 200 μl) in U-bottom 96-well plates.After a six hour incubation period at 37° C., a 0.1 ml aliquot ofsupernatant is removed from each well and radioactivity is determined ina Micromedic automatic gamma counter. The percent specific lysis isdetermined by the formula: percent specific release=100×(experimentalrelease−spontaneous release)/(maximum release−spontaneous release). Tofacilitate comparison between separate CTL assays run under the sameconditions, % ⁵¹Cr release data is expressed as lytic units/10⁶ cells.One lytic unit is arbitrarily defined as the number of effector cellsrequired to achieve 30% lysis of 10,000 target cells in a six hour ⁵¹Crrelease assay. To obtain specific lytic units/10⁶, the lytic units/10⁶obtained in the absence of peptide is subtracted from the lyticunits/10⁶ obtained in the presence of peptide. For example, if 30% ⁵¹Crrelease is obtained at the effector (E): target (T) ratio of 50:1 (i.e.,5×10⁵ effector cells for 10,000 targets) in the absence of peptide and5:1 (i.e., 5×10⁴ effector cells for 10,000 targets) in the presence ofpeptide, the specific lytic units would be:[(1/50,000)−(1/500,000)]×10⁶=18 LU.

The results are analyzed to assess the magnitude of the CTL responses ofanimals injected with the immunogenic CTL/HTL conjugate vaccinepreparation and are compared to the magnitude of the CTL responseachieved using, for example, CTL epitopes as outlined above in theExample entitled “Confirmation of Immunogenicity.” Analyses similar tothis may be performed to confirm the immunogenicity of peptideconjugates containing multiple CTL epitopes and/or multiple HTLepitopes. In accordance with these procedures, it is found that a CTLresponse is induced, and concomitantly that an HTL response is inducedupon administration of such compositions.

Example 21 Selection of CTL and HTL Epitopes for Inclusion in a24P4C12-Specific Vaccine

This example illustrates a procedure for selecting peptide epitopes forvaccine compositions of the invention. The peptides in the compositioncan be in the form of a nucleic acid sequence, either single or one ormore sequences (i.e., minigene) that encodes peptide(s), or can besingle and/or polyepitopic peptides.

The following principles are utilized when selecting a plurality ofepitopes for inclusion in a vaccine composition. Each of the followingprinciples is balanced in order to make the selection.

Epitopes are selected which, upon administration, mimic immune responsesthat are correlated with 24P4C12 clearance. The number of epitopes useddepends on observations of patients who spontaneously clear 24P4C12. Forexample, if it has been observed that patients who spontaneously clear24P4C12-expressing cells generate an immune response to at least three(3) epitopes from 24P4C12 antigen, then at least three epitopes shouldbe included for HLA class I. A similar rationale is used to determineHLA class II epitopes.

Epitopes are often selected that have a binding affinity of an IC₅₀ of500 nM or less for an HLA class I molecule, or for class II, an IC₅₀ of1000 nM or less; or HLA Class I peptides with high binding scores fromthe BIMAS web site, at URL bimas.dcrt.nih.gov/.

In order to achieve broad coverage of the vaccine through out a diversepopulation, sufficient supermotif bearing peptides, or a sufficientarray of allele-specific motif bearing peptides, are selected to givebroad population coverage. In one embodiment, epitopes are selected toprovide at least 80% population coverage. A Monte Carlo analysis, astatistical evaluation known in the art, can be employed to assessbreadth, or redundancy, of population coverage.

When creating polyepitopic compositions, or a minigene that encodessame, it is typically desirable to generate the smallest peptidepossible that encompasses the epitopes of interest. The principlesemployed are similar, if not the same, as those employed when selectinga peptide comprising nested epitopes. For example, a protein sequencefor the vaccine composition is selected because it has maximal number ofepitopes contained within the sequence, i.e., it has a highconcentration of epitopes. Epitopes may be nested or overlapping (i.e.,frame shifted relative to one another). For example, with overlappingepitopes, two 9-mer epitopes and one 10-mer epitope can be present in a10 amino acid peptide. Each epitope can be exposed and bound by an HLAmolecule upon administration of such a peptide. A multi-epitopic,peptide can be generated synthetically, recombinantly, or via cleavagefrom the native source. Alternatively, an analog can be made of thisnative sequence, whereby one or more of the epitopes comprisesubstitutions that alter the cross-reactivity and/or binding affinityproperties of the polyepitopic peptide. Such a vaccine composition isadministered for therapeutic or prophylactic purposes. This embodimentprovides for the possibility that an as yet undiscovered aspect ofimmune system processing will apply to the native nested sequence andthereby facilitate the production of therapeutic or prophylactic immuneresponse-inducing vaccine compositions. Additionally such an embodimentprovides for the possibility of motif-bearing epitopes for an HLA makeupthat is presently unknown. Furthermore, this embodiment (absent thecreating of any analogs) directs the immune response to multiple peptidesequences that are actually present in 24P4C12, thus avoiding the needto evaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing nucleic acid vaccine compositions.Related to this embodiment, computer programs can be derived inaccordance with principles in the art, which identify in a targetsequence, the greatest number of epitopes per sequence length.

A vaccine composition comprised of selected peptides, when administered,is safe, efficacious, and elicits an immune response similar inmagnitude to an immune response that controls or clears cells that bearor overexpress 24P4C12.

Example 22 Construction of “Minigene” Multi-Epitope DNA Plasmids

This example discusses the construction of a minigene expressionplasmid. Minigene plasmids may, of course, contain variousconfigurations of B cell, CTL and/or HTL epitopes or epitope analogs asdescribed herein.

A minigene expression plasmid typically includes multiple CTL and HTLpeptide epitopes. In the present example, HLA-A2, -A3, -B7supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearingpeptide epitopes are used in conjunction with DR supermotif-bearingepitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearingpeptide epitopes derived 24P4C12, are selected such that multiplesupermotifs/motifs are represented to ensure broad population coverage.Similarly, HLA class II epitopes are selected from 24P4C12 to providebroad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearingepitopes and HLA DR-3 motif-bearing epitopes are selected for inclusionin the minigene construct. The selected CTL and HTL epitopes are thenincorporated into a minigene for expression in an expression vector.

Such a construct may additionally include sequences that direct the HTLepitopes to the endoplasmic reticulum. For example, the Ii protein maybe fused to one or more HTL epitopes as described in the art, whereinthe CLIP sequence of the Ii protein is removed and replaced with an HLAclass II epitope sequence so that HLA class II epitope is directed tothe endoplasmic reticulum, where the epitope binds to an HLA class IImolecules.

This example illustrates the methods to be used for construction of aminigene-bearing expression plasmid. Other expression vectors that maybe used for minigene compositions are available and known to those ofskill in the art.

The minigene DNA plasmid of this example contains a consensus Kozaksequence and a consensus murine kappa Ig-light chain signal sequencefollowed by CTL and/or HTL epitopes selected in accordance withprinciples disclosed herein. The sequence encodes an open reading framefused to the Myc and H is antibody epitope tag coded for by the pcDNA3.1 Myc-His vector.

Overlapping oligonucleotides that can, for example, average about 70nucleotides in length with 15 nucleotide overlaps, are synthesized andHPLC-purified. The oligonucleotides encode the selected peptide epitopesas well as appropriate linker nucleotides, Kozak sequence, and signalsequence. The final multiepitope minigene is assembled by extending theoverlapping oligonucleotides in three sets of reactions using PCR. APerkin/Elmer 9600 PCR machine is used and a total of 30 cycles areperformed using the following conditions: 95° C. for 15 sec, annealingtemperature (5° below the lowest calculated Tm of each primer pair) for30 sec, and 72° C. for 1 min.

For example, a minigene is prepared as follows. For a first PCRreaction, 5 μg of each of two oligonucleotides are annealed andextended: In an example using eight oligonucleotides, i.e., four pairsof primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100μl reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM(NH4)₂SO₄, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO₄, 0.1% Triton X-100,100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. Thefull-length dimer products are gel-purified, and two reactionscontaining the product of 1+2 and 3+4, and the product of 5+6 and 7+8are mixed, annealed, and extended for 10 cycles. Half of the tworeactions are then mixed, and 5 cycles of annealing and extensioncarried out before flanking primers are added to amplify the full lengthproduct. The full-length product is gel-purified and cloned intopCR-blunt (Invitrogen) and individual clones are screened by sequencing.

Example 23 The Plasmid Construct and the Degree to which it InducesImmunogenicity

The degree to which a plasmid construct, for example a plasmidconstructed in accordance with the previous Example, is able to induceimmunogenicity is confirmed in vitro by determining epitope presentationby APC following transduction or transfection of the APC with anepitope-expressing nucleic acid construct. Such a study determines“antigenicity” and allows the use of human APC. The assay determines theability of the epitope to be presented by the APC in a context that isrecognized by a T cell by quantifying the density of epitope-HLA class Icomplexes on the cell surface. Quantitation can be performed by directlymeasuring the amount of peptide eluted from the APC (see, e.g., Sijts etal., J. Immunol. 156:683-692, 1996; Demotz et al., Nature 342:682-684,1989); or the number of peptide-HLA class I complexes can be estimatedby measuring the amount of lysis or lymphokine release induced bydiseased or transfected target cells, and then determining theconcentration of peptide necessary to obtain equivalent levels of lysisor lymphokine release (see, e.g., Kageyama et al., J. Immunol154:567-576, 1995).

Alternatively, immunogenicity is confirmed through in vivo injectionsinto mice and subsequent in vitro assessment of CTL and HTL activity,which are analyzed using cytotoxicity and proliferation assays,respectively, as detailed e.g., in Alexander et al., Immunity 1:751-761,1994.

For example, to confirm the capacity of a DNA minigene constructcontaining at least one HLA-A2 supermotif peptide to induce CTLs invivo, HLA-A2.1/K^(b) transgenic mice, for example, are immunizedintramuscularly with 100 μg of naked cDNA. As a means of comparing thelevel of CTLs induced by cDNA immunization, a control group of animalsis also immunized with an actual peptide composition that comprisesmultiple epitopes synthesized as a single polypeptide as they would beencoded by the minigene.

Splenocytes from immunized animals are stimulated twice with each of therespective compositions (peptide epitopes encoded in the minigene or thepolyepitopic peptide), then assayed for peptide-specific cytotoxicactivity in a ⁵¹Cr release assay. The results indicate the magnitude ofthe CTL response directed against the A2-restricted epitope, thusindicating the in vivo immunogenicity of the minigene vaccine andpolyepitopic vaccine.

It is, therefore, found that the minigene elicits immune responsesdirected toward the HLA-A2 supermotif peptide epitopes as does thepolyepitopic peptide vaccine. A similar analysis is also performed usingother HLA-A3 and HLA-B7 transgenic mouse models to assess CTL inductionby HLA-A3 and HLA-B7 motif or supermotif epitopes, whereby it is alsofound that the minigene elicits appropriate immune responses directedtoward the provided epitopes.

To confirm the capacity of a class II epitope-encoding minigene toinduce HTLs in vivo, DR transgenic mice, or for those epitopes thatcross react with the appropriate mouse MHC molecule, I-Ab-restrictedmice, for example, are immunized intramuscularly with 100 μg of plasmidDNA. As a means of comparing the level of HTLs induced by DNAimmunization, a group of control animals is also immunized with anactual peptide composition emulsified in complete Freund's adjuvant.CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunizedanimals and stimulated with each of the respective compositions(peptides encoded in the minigene). The HTL response is measured using a³H-thymidine incorporation proliferation assay, (see, e.g., Alexander etal. Immunity 1:751-761, 1994). The results indicate the magnitude of theHTL response, thus demonstrating the in vivo immunogenicity of theminigene.

DNA minigenes, constructed as described in the previous Example, canalso be confirmed as a vaccine in combination with a boosting agentusing a prime boost protocol. The boosting agent can consist ofrecombinant protein (e.g., Barnett et al., Aids Res. and HumanRetroviruses 14, Supplement 3:S299-S309,1998) or recombinant vaccinia,for example, expressing a minigene or DNA encoding the complete proteinof interest (see, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegahet al., Proc. Natl. Acad. Sci. USA 95:7648-53, 1998; Hanke andMcMichael, Immunol. Letters 66:177-181, 1999; and Robinson et al.,Nature Med. 5:526-34, 1999).

For example, the efficacy of the DNA minigene used in a prime boostprotocol is initially evaluated in transgenic mice. In this example,A2.1/K^(b) transgenic mice are immunized IM with 100 μg of a DNAminigene encoding the immunogenic peptides including at least one HLA-A2supermotif-bearing peptide. After an incubation period (ranging from 3-9weeks), the mice are boosted IP with 10⁷ pfu/mouse of a recombinantvaccinia virus expressing the same sequence encoded by the DNA minigene.Control mice are immunized with 100 μg of DNA or recombinant vacciniawithout the minigene sequence, or with DNA encoding the minigene, butwithout the vaccinia boost. After an additional incubation period of twoweeks, splenocytes from the mice are immediately assayed forpeptide-specific activity in an ELISPOT assay. Additionally, splenocytesare stimulated in vitro with the A2-restricted peptide epitopes encodedin the minigene and recombinant vaccinia, then assayed forpeptide-specific activity in an alpha, beta and/or gamma IFN ELISA.

It is found that the minigene utilized in a prime-boost protocol elicitsgreater immune responses toward the HLA-A2 supermotif peptides than withDNA alone. Such an analysis can also be performed using HLA-A11 orHLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 orHLA-B7 motif or supermotif epitopes. The use of prime boost protocols inhumans is described below in the Example entitled “Induction of CTLResponses Using a Prime Boost Protocol.”

Example 24 Peptide Compositions for Prophylactic Uses

Vaccine compositions of the present invention can be used to prevent24P4C12 expression in persons who are at risk for tumors that bear thisantigen. For example, a polyepitopic peptide epitope composition (or anucleic acid comprising the same) containing multiple CTL and HTLepitopes such as those selected in the above Examples, which are alsoselected to target greater than 80% of the population, is administeredto individuals at risk for a 24P4C12-associated tumor.

For example, a peptide-based composition is provided as a singlepolypeptide that encompasses multiple epitopes. The vaccine is typicallyadministered in a physiological solution that comprises an adjuvant,such as Incomplete Freunds Adjuvant. The dose of peptide for the initialimmunization is from about 1 to about 50,000 g, generally 100-5,000 μg,for a 70 kg patient. The initial administration of vaccine is followedby booster dosages at 4 weeks followed by evaluation of the magnitude ofthe immune response in the patient, by techniques that determine thepresence of epitope-specific CTL populations in a PBMC sample.Additional booster doses are administered as required. The compositionis found to be both safe and efficacious as a prophylaxis against24P4C12-associated disease.

Alternatively, a composition typically comprising transfecting agents isused for the administration of a nucleic acid-based vaccine inaccordance with methodologies known in the art and disclosed herein.

Example 25 Polyepitopic Vaccine Compositions Derived from Native 24P4C12Sequences

A native 24P4C12 polyprotein sequence is analyzed, preferably usingcomputer algorithms defined for each class I and/or class II supermotifor motif, to identify “relatively short” regions of the polyprotein thatcomprise multiple epitopes. The “relatively short” regions arepreferably less in length than an entire native antigen. This relativelyshort sequence that contains multiple distinct or overlapping, “nested”epitopes can be used to generate a minigene construct. The construct isengineered to express the peptide, which corresponds to the nativeprotein sequence. The “relatively short” peptide is generally less than250 amino acids in length, often less than 100 amino acids in length,preferably less than 75 amino acids in length, and more preferably lessthan 50 amino acids in length. The protein sequence of the vaccinecomposition is selected because it has maximal number of epitopescontained within the sequence, i.e., it has a high concentration ofepitopes. As noted herein, epitope motifs may be nested or overlapping(i.e., frame shifted relative to one another). For example, withoverlapping epitopes, two 9-mer epitopes and one 10-mer epitope can bepresent in a 10 amino acid peptide. Such a vaccine composition isadministered for therapeutic or prophylactic purposes.

The vaccine composition will include, for example, multiple CTL epitopesfrom 24P4C12 antigen and at least one HTL epitope. This polyepitopicnative sequence is administered either as a peptide or as a nucleic acidsequence which encodes the peptide. Alternatively, an analog can be madeof this native sequence, whereby one or more of the epitopes comprisesubstitutions that alter the cross-reactivity and/or binding affinityproperties of the polyepitopic peptide.

The embodiment of this example provides for the possibility that an asyet undiscovered aspect of immune system processing will apply to thenative nested sequence and thereby facilitate the production oftherapeutic or prophylactic immune response-inducing vaccinecompositions. Additionally, such an embodiment provides for thepossibility of motif-bearing epitopes for an HLA makeup(s) that ispresently unknown. Furthermore, this embodiment (excluding an analogedembodiment) directs the immune response to multiple peptide sequencesthat are actually present in native 24P4C12, thus avoiding the need toevaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing peptide or nucleic acid vaccinecompositions.

Related to this embodiment, computer programs are available in the artwhich can be used to identify in a target sequence, the greatest numberof epitopes per sequence length.

Example 26 Polyepitopic Vaccine Compositions from Multiple Antigens

The 24P4C12 peptide epitopes of the present invention are used inconjunction with epitopes from other target tumor-associated antigens,to create a vaccine composition that is useful for the prevention ortreatment of cancer that expresses 24P4C12 and such other antigens. Forexample, a vaccine composition can be provided as a single polypeptidethat incorporates multiple epitopes from 24P4C12 as well astumor-associated antigens that are often expressed with a target cancerassociated with 24P4C12 expression, or can be administered as acomposition comprising a cocktail of one or more discrete epitopes.Alternatively, the vaccine can be administered as a minigene constructor as dendritic cells which have been loaded with the peptide epitopesin vitro.

Example 27 Use of Peptides to Evaluate an Immune Response

Peptides of the invention may be used to analyze an immune response forthe presence of specific antibodies, CTL or HTL directed to 24P4C12.Such an analysis can be performed in a manner described by Ogg et al.,Science 279:2103-2106,1998. In this Example, peptides in accordance withthe invention are used as a reagent for diagnostic or prognosticpurposes, not as an immunogen.

In this example highly sensitive human leukocyte antigen tetramericcomplexes (“tetramers”) are used for a cross-sectional analysis of, forexample, 24P4C12 HLA-A*0201-specific CTL frequencies from HLAA*0201-positive individuals at different stages of disease or followingimmunization comprising a 24P4C12 peptide containing an A*0201 motif.Tetrameric complexes are synthesized as described (Musey et al., N.Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201in this example) and β2-microglobulin are synthesized by means of aprokaryotic expression system. The heavy chain is modified by deletionof the transmembrane-cytosolic tail and COOH-terminal addition of asequence containing a BirA enzymatic biotinylation site. The heavychain, β2-microglobulin, and peptide are refolded by dilution. The 45-kDrefolded product is isolated by fast protein liquid chromatography andthen biotinylated by BirA in the presence of biotin (Sigma, St. Louis,Mo.), adenosine 5′ triphosphate and magnesium.Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, andthe tetrameric product is concentrated to 1 mg/ml. The resulting productis referred to as tetramer-phycoerythrin.

For the analysis of patient blood samples, approximately one millionPBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 μl ofcold phosphate-buffered saline. Tri-color analysis is performed with thetetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. ThePBMCs are incubated with tetramer and antibodies on ice for 30 to 60 minand then washed twice before formaldehyde fixation. Gates are applied tocontain >99.98% of control samples. Controls for the tetramers includeboth A*0201-negative individuals and A*0201-positive non-diseaseddonors. The percentage of cells stained with the tetramer is thendetermined by flow cytometry. The results indicate the number of cellsin the PBMC sample that contain epitope-restricted CTLs, thereby readilyindicating the extent of immune response to the 24P4C12 epitope, andthus the status of exposure to 24P4C12, or exposure to a vaccine thatelicits a protective or therapeutic response.

Example 28 Use of Peptide Epitopes to Evaluate Recall Responses

The peptide epitopes of the invention are used as reagents to evaluate Tcell responses, such as acute or recall responses, in patients. Such ananalysis may be performed on patients who have recovered from24P4C12-associated disease or who have been vaccinated with a 24P4C12vaccine.

For example, the class I restricted CTL response of persons who havebeen vaccinated may be analyzed. The vaccine may be any 24P4C12 vaccine.PBMC are collected from vaccinated individuals and HLA typed.Appropriate peptide epitopes of the invention that, optimally, bearsupermotifs to provide cross-reactivity with multiple HLA supertypefamily members, are then used for analysis of samples derived fromindividuals who bear that HLA type.

PBMC from vaccinated individuals are separated on Ficoll-Histopaquedensity gradients (Sigma Chemical Co., St. Louis, Mo.), washed threetimes in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCOLaboratories) supplemented with L-glutamine (2 mM), penicillin (50U/ml), streptomycin (50 μg/ml), and Hepes (10 mM) containing 10%heat-inactivated human AB serum (complete RPMI) and plated usingmicroculture formats. A synthetic peptide comprising an epitope of theinvention is added at 10 μg/ml to each well and HBV core 128-140 epitopeis added at 1 μg/ml to each well as a source of T cell help during thefirst week of stimulation.

In the microculture format, 4×10⁵ PBMC are stimulated with peptide in 8replicate cultures in 96-well round bottom plate in 100 μl/well ofcomplete RPMI. On days 3 and 10,100 μl of complete RPMI and 20 U/mlfinal concentration of rIL-2 are added to each well. On day 7 thecultures are transferred into a 96-well flat-bottom plate andrestimulated with peptide, rIL-2 and 10⁵ irradiated (3,000 rad)autologous feeder cells. The cultures are tested for cytotoxic activityon day 14. A positive CTL response requires two or more of the eightreplicate cultures to display greater than 10% specific ⁵¹Cr release,based on comparison with non-diseased control subjects as previouslydescribed (Rehermann, et al., Nature Med. 2:1104,1108,1996; Rehermann etal., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J. Clin.Invest. 98:1432-1440, 1996).

Target cell lines are autologous and allogeneic EBV-transformed B-LCLthat are either purchased from the American Society forHistocompatibility and Immunogenetics (ASHI, Boston, Mass.) orestablished from the pool of patients as described (Guilhot, et al. J.Virol. 66:2670-2678, 1992).

Cytotoxicity assays are performed in the following manner. Target cellsconsist of either allogeneic HLA-matched or autologous EBV-transformed Blymphoblastoid cell line that are incubated overnight with the syntheticpeptide epitope of the invention at 10 μM, and labeled with 100 μCi of⁵¹Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after whichthey are washed four times with HBSS.

Cytolytic activity is determined in a standard 4-h, split well ⁵¹Crrelease assay using U-bottomed 96 well plates containing 3,000targets/well. Stimulated PBMC are tested at effector/target (E/T) ratiosof 20-50:1 on day 14. Percent cytotoxicity is determined from theformula: 100×[(experimental release−spontaneous release)/maximumrelease−spontaneous release)]. Maximum release is determined by lysis oftargets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis,Mo.). Spontaneous release is <25% of maximum release for allexperiments.

The results of such an analysis indicate the extent to whichHLA-restrioted CTL populations have been stimulated by previous exposureto 24P4C12 or a 24P4C12 vaccine.

Similarly, Class II restricted HTL responses may also be analyzed.Purified PBMC are cultured in a 96-well flat bottom plate at a densityof 1.5×10⁵ cells/well and are stimulated with 10 μg/ml synthetic peptideof the invention, whole 24P4C12 antigen, or PHA. Cells are routinelyplated in replicates of 4-6 wells for each condition. After seven daysof culture, the medium is removed and replaced with fresh mediumcontaining 10 U/ml IL-2. Two days later, 1 μCi ³H-thymidine is added toeach well and incubation is continued for an additional 18 hours.Cellular DNA is then harvested on glass fiber mats and analyzed for³H-thymidine incorporation. Antigen-specific T cell proliferation iscalculated as the ratio of 3H-thymidine incorporation in the presence ofantigen divided by the 3H-thymidine incorporation in the absence ofantigen.

Example 29 Induction of Specific CTL Response in Humans

A human clinical trial for an immunogenic composition comprising CTL andHTL epitopes of the invention is set up as an IND Phase I, doseescalation study and carried out as a randomized, double-blind,placebo-controlled trial. Such a trial is designed, for example, asfollows:

A total of about 27 individuals are enrolled and divided into 3 groups:

Group I: 3 subjects are injected with placebo and 6 subjects areinjected with 5 μg of peptide composition;

Group II: 3 subjects are injected with placebo and 6 subjects areinjected with 50 μg peptide composition;

Group III: 3 subjects are injected with placebo and 6 subjects areinjected with 500 μg of peptide composition.

After 4 weeks following the first injection, all subjects receive abooster inoculation at the same dosage.

The endpoints measured in this study relate to the safety andtolerability of the peptide composition as well as its immunogenicity.Cellular immune responses to the peptide composition are an index of theintrinsic activity of this the peptide composition, and can therefore beviewed as a measure of biological efficacy. The following summarize theclinical and laboratory data that relate to safety and efficacyendpoints.

Safety: The incidence of adverse events is monitored in the placebo anddrug treatment group and assessed in terms of degree and reversibility.

Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy,subjects are bled before and after injection. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

The vaccine is found to be both safe and efficacious.

Example 30 Phase II Trials in Patients Expressing 24P4C12

Phase II trials are performed to study the effect of administering theCTL-HTL peptide compositions to patients having cancer that expresses24P4C12. The main objectives of the trial are to determine an effectivedose and regimen for inducing CTLs in cancer patients that express24P4C12, to establish the safety of inducing a CTL and HTL response inthese patients, and to see to what extent activation of CTLs improvesthe clinical picture of these patients, as manifested, e.g., by thereduction and/or shrinking of lesions. Such a study is designed, forexample, as follows:

The studies are performed in multiple centers. The trial design is anopen-label, uncontrolled, dose escalation protocol wherein the peptidecomposition is administered as a single dose followed six weeks later bya single booster shot of the same dose. The dosages are 50, 500 and5,000 micrograms per injection. Drug-associated adverse effects(severity and reversibility) are recorded.

There are three patient groupings. The first group is injected with 50micrograms of the peptide composition and the second and third groupswith 500 and 5,000 micrograms of peptide composition, respectively. Thepatients within each group range in age from 21-65 and represent diverseethnic backgrounds. All of them have a tumor that expresses 24P4C12.

Clinical manifestations or antigen-specific T-cell responses aremonitored to assess the effects of administering the peptidecompositions. The vaccine composition is found to be both safe andefficacious in the treatment of 24P4C12-associated disease.

Example 31 Induction of CTL Responses Using a Prime Boost Protocol

A prime boost protocol similar in its underlying principle to that usedto confirm the efficacy of a DNA vaccine in transgenic mice, such asdescribed above in the Example entitled “The Plasmid Construct and theDegree to Which It Induces Immunogenicity,” can also be used for theadministration of the vaccine to humans. Such a vaccine regimen caninclude an initial administration of, for example, naked DNA followed bya boost using recombinant virus encoding the vaccine, or recombinantprotein/polypeptide or a peptide mixture administered in an adjuvant.

For example, the initial immunization may be performed using anexpression vector, such as that constructed in the Example entitled“Construction of “Minigene” Multi-Epitope DNA Plasmids” in the form ofnaked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also beadministered using a gene gun. Following an incubation period of 3-4weeks, a booster dose is then administered. The booster can berecombinant fowlpox virus administered at a dose of 5-10⁷ to 5×10⁹ pfu.An alternative recombinant virus, such as an MVA, canarypox, adenovirus,or adeno-associated virus, can also be used for the booster, or thepolyepitopic protein or a mixture of the peptides can be administered.For evaluation of vaccine efficacy, patient blood samples are obtainedbefore immunization as well as at intervals following administration ofthe initial vaccine and booster doses of the vaccine. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

Analysis of the results indicates that a magnitude of responsesufficient to achieve a therapeutic or protective immunity against24P4C12 is generated.

Example 32 Administration of Vaccine Compositions Using Dendritic Cells(DC)

Vaccines comprising peptide epitopes of the invention can beadministered using APCs, or “professional” APCs such as DC. In thisexample, peptide-pulsed DC are administered to a patient to stimulate aCTL response in vivo. In this method, dendritic cells are isolated,expanded, and pulsed with a vaccine comprising peptide CTL and HTLepitopes of the invention. The dendritic cells are infused back into thepatient to elicit CTL and HTL responses in vivo. The induced CTL and HTLthen destroy or facilitate destruction, respectively, of the targetcells that bear the 24P4C12 protein from which the epitopes in thevaccine are derived.

For example, a cocktail of epitope-comprising peptides is administeredex vivo to PBMC, or isolated DC therefrom. A pharmaceutical tofacilitate harvesting of DC can be used, such as Progenipoietin™(Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC withpeptides, and prior to reinfusion into patients, the DC are washed toremove unbound peptides.

As appreciated clinically, and readily determined by one of skill basedon clinical outcomes, the number of DC reinfused into the patient canvary (see, e.g., Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 andProstate 32:272, 1997). Although 2-50×10⁶ DC per patient are typicallyadministered, larger number of DC, such as 10⁷ or 10⁸ can also beprovided. Such cell populations typically contain between 50-90% DC.

In some embodiments, peptide-loaded PBMC are injected into patientswithout purification of the DC. For example, PBMC generated aftertreatment with an agent such as Progenipoietin™ are injected intopatients without purification of the DC. The total number of PBMC thatare administered often ranges from 10⁸ to 10¹⁰. Generally, the celldoses injected into patients is based on the percentage of DC in theblood of each patient, as determined, for example, by immunofluorescenceanalysis with specific anti-DC antibodies. Thus, for example, ifProgenipoietin™ mobilizes 2% DC in the peripheral blood of a givenpatient, and that patient is to receive 5×10⁶ DC, then the patient willbe injected with a total of 2.5×10⁸ peptide-loaded PBMC. The percent DCmobilized by an agent such as Progenipoietin™ is typically estimated tobe between 2-10%, but can vary as appreciated by one of skill in theart.

Ex Vivo Activation of CTL/HTL Responses

Alternatively, ex vivo CTL or HTL responses to 24P4C12 antigens can beinduced by incubating, in tissue culture, the patient's, or geneticallycompatible, CTL or HTL precursor cells together with a source of APC,such as DC, and immunogenic peptides. After an appropriate incubationtime (typically about 7-28 days), in which the precursor cells areactivated and expanded into effector cells, the cells are infused intothe patient, where they will destroy (CTL) or facilitate destruction(HTL) of their specific target cells, i.e., tumor cells.

Example 33 An Alternative Method of Identifying and ConfirmingMotif-Bearing Peptides

Another method of identifying and confirming motif-bearing peptides isto elute them from cells bearing defined MHC molecules. For example, EBVtransformed B cell lines used for tissue typing have been extensivelycharacterized to determine which HLA molecules they express. In certaincases these cells express only a single type of HLA molecule. Thesecells can be transfected with nucleic acids that express the antigen ofinterest, e.g. 24P4C12. Peptides produced by endogenous antigenprocessing of peptides produced as a result of transfection will thenbind to HLA molecules within the cell and be transported and displayedon the cell's surface. Peptides are then eluted from the HLA moleculesby exposure to mild acid conditions and their amino acid sequencedetermined, e.g., by mass spectral analysis (e.g., Kubo et al., J.Immunol. 152:3913, 1994). Because the majority of peptides that bind aparticular HLA molecule are motif-bearing, this is an alternativemodality for obtaining the motif-bearing peptides correlated with theparticular HLA molecule expressed on the cell.

Alternatively, cell lines that do not express endogenous HLA moleculescan be transfected with an expression construct encoding a single HLAallele. These cells can then be used as described, i.e., they can thenbe transfected with nucleic acids that encode 24P4C12 to isolatepeptides corresponding to 24P4C12 that have been presented on the cellsurface. Peptides obtained from such an analysis will bear motif(s) thatcorrespond to binding to the single HLA allele that is expressed in thecell.

As appreciated by one in the art, one can perform a similar analysis ona cell bearing more than one HLA allele and subsequently determinepeptides specific for each HLA allele expressed. Moreover, one of skillwould also recognize that means other than transfection, such as loadingwith a protein antigen, can be used to provide a source of antigen tothe cell.

Example 34 Complementary Polynucleotides

Sequences complementary to the 24P4C12-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring 24P4C12. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06software (National Biosciences) and the coding sequence of 24P4C12. Toinhibit transcription, a complementary oligonucleotide is designed fromthe most unique 5′ sequence and used to prevent promoter binding to thecoding sequence. To inhibit translation, a complementary oligonucleotideis designed to prevent ribosomal binding to a 24P4C12-encodingtranscript.

Example 35 Purification of Naturally-occurring or Recombinant 24P4C12Using 24P4C12-Specific Antibodies

Naturally occurring or recombinant 24P4C12 is substantially purified byimmunoaffinity chromatography using antibodies specific for 24P4C12. Animmunoaffinity column is constructed by covalently coupling anti-24P4C12antibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin isblocked and washed according to the manufacturer's instructions.

Media containing 24P4C12 are passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of 24P4C12 (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/24P4C12 binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andGCR.P is collected.

Example 36 Identification of Molecules which Interact with 24P4C12

24P4C12, or biologically active fragments thereof, are labeled with 1211 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled 24P4C12, washed, and anywells with labeled 24P4C12 complex are assayed. Data obtained usingdifferent concentrations of 24P4C12 are used to calculate values for thenumber, affinity, and association of 24P4C12 with the candidatemolecules.

Example 37 In Vivo Assay for 24P4C12 Tumor Growth Promotion

The effect of the 24P4C12 protein on tumor cell growth is evaluated invivo by evaluating tumor development and growth of cells expressing orlacking 24P4C12. For example, SCID mice are injected subcutaneously oneach flank with 1×10⁶ of either 3T3, prostate, colon, ovary, lung, orbladder cancer cell lines (e.g. PC3, Caco, PA-1, CaLu or J82 cells)containing tkNeo empty vector or 24P4C12. At least two strategies may beused: (1) Constitutive 24P4C12 expression under regulation of apromoter, such as a constitutive promoter obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virusand Simian Virus 40 (SV40), or from heterologous mammalian promoters,e.g., the actin promoter or an immunoglobulin promoter, provided suchpromoters are compatible with the host cell systems, and (2) Regulatedexpression under control of an inducible vector system, such asecdysone, tetracycline, etc., provided such promoters are compatiblewith the host cell systems. Tumor volume is then monitored by calipermeasurement at the appearance of palpable tumors and followed over timeto determine if 24P4C12-expressing cells grow at a faster rate andwhether tumors produced by 24P4C12-expressing cells demonstratecharacteristics of altered aggressiveness (e.g. enhanced metastasis,vascularization, reduced responsiveness to chemotherapeutic drugs). Asshown in FIG. 31 and FIG. 32, 24P4C12 has a profound effect on tumorgrowth in SCID mice. The prostate cancer cells PC3 and PC3-24P4C12 wereinjected subcutaneously in the right flank of SCID mice. Tumor growthwas evaluated by caliper measurements. An increase in tumor growth wasobserved in PC3-24P4C12 tumors within 47 days of injection (FIG. 31). Inaddition, subcutaneous injection of 3T3-24P4C12 induced tumor formationin SCID mice (FIG. 32). This finding is significant as control 3T3 cellsfail to form tumors, indicating that 24P4C12 has several tumor enhancingcapabilities, including transformation, as well as tumor initiation andpromotion.

Example 38 24P4C12 Monoclonal Antibody-mediated Inhibition of ProstateTumors In Vivo

The significant expression of 24P4C12 in cancer tissues, together withits restrictive expression in normal tissues and cell surfacelocalization, make 24P4C12 a good target for antibody therapy.Similarly, 24P4C12 is a target for T cell-based immunotherapy. Thus, thetherapeutic efficacy of anti-24P4C12 mAbs in human prostate cancerxenograft mouse models is evaluated by using recombinant cell lines suchas PC3-24P4C12, and 3T3-24P4C12 (see, e.g., Kaighn, M. E., et al.,Invest Urol, 1979. 17(1): p. 16-23), as well as human prostate xenograftmodels such as LAPC9 (Saffran et al., Proc Natl Acad Sci USA. 2001,98:2658). Similarly, anti-24P4C12 mAbs are evaluated in xenograft modelsof human bladder cancer colon cancer, ovarian cancer or lung cancerusing recombinant cell lines such as J82-24P4C12, Caco-24P4C12,PA-24P4C1 or CaLu-24P4C12, respectively.

Antibody efficacy on tumor growth and metastasis formation is studied,e.g., in a mouse orthotopic bladder cancer xenograft model, and a mouseprostate cancer xenograft model. The antibodies can be unconjugated, asdiscussed in this Example, or can be conjugated to a therapeuticmodality, as appreciated in the art. Anti-24P4C12 mAbs inhibit formationof prostate and bladder xenografts. Anti-24P4C12 mAbs also retard thegrowth of established orthotopic tumors and prolonged survival oftumor-bearing mice. These results indicate the utility of anti-24P4C12mAbs in the treatment of local and advanced stages of prostate, colon,ovarian, lung and bladder cancer. (See, e.g., Saffran, D., et al., PNAS10:1073-1078 or www.pnas.org/cgi/doi/10.1073/pnas.051624698).

Administration of the anti-24P4C12 mAbs led to retardation ofestablished orthotopic tumor growth and inhibition of metastasis todistant sites, resulting in a significant prolongation in the survivalof tumor-bearing mice. These studies indicate that 24P4C12 as anattractive target for immunotherapy and demonstrate the therapeuticpotential of anti-24P4C12 mAbs for the treatment of local and metastaticcancer. This example demonstrates that unconjugated 24P4C12 monoclonalantibodies are effective to inhibit the growth of human prostate, colon,ovarian, lung and bladder cancer tumor xenografts grown in SCID mice;accordingly a combination of such efficacious monoclonal antibodies isalso effective.

Tumor Inhibition using Multiple Unconjugated 24P4C12 mAbs

Materials and Methods

24P4C12 Monoclonal Antibodies:

Monoclonal antibodies are raised against 24P4C12 as described in theExample entitled “Generation of 24P4C12 Monoclonal Antibodies (mAbs).”The antibodies are characterized by ELISA, Western blot, FACS, andimmunoprecipitation for their capacity to bind 24P4C12. Epitope mappingdata for the anti-24P4C12 mAbs, as determined by ELISA and Westernanalysis, recognize epitopes on the 24P4C12 protein. Immunohistochemicalanalysis of prostate cancer tissues and cells with these antibodies isperformed.

The monoclonal antibodies are purified from ascites or hybridoma tissueculture supernatants by Protein-G Sepharose chromatography, dialyzedagainst PBS, filter sterilized, and stored at −20° C. Proteindeterminations are performed by a Bradford assay (Bio-Rad, Hercules,Calif.). A therapeutic monoclonal antibody or a cocktail comprising amixture of individual monoclonal antibodies is prepared and used for thetreatment of mice receiving subcutaneous or orthotopic injections ofSCABER, J82, A498, 769P, CaOv1 or PA1 tumor xenografts.

Cell Lines

The prostate, colon, ovarian, lung and bladder cancer carcinoma celllines, Caco, PA-1, CaLu or J82 cells as well as the fibroblast line NIH3T3 (American Type Culture Collection) are maintained in mediasupplemented with L-glutamine and 10% FBS.

PC3-24P4C12, Caco-24P4C12, PA-24P4C12, CaLu-24P4C12 or J82-24P4C12 cellsand 3T3-24P4C12 cell populations are generated by retroviral genetransfer as described in Hubert, R. S., et al., Proc Natl Acad Sci USA,1999. 96(25): 14523.

Xenograft Mouse Models.

Subcutaneous (s.c.) tumors are generated by injection of 1×10⁶ cancercells mixed at a 1:1 dilution with Matrigel (Collaborative Research) inthe right flank of male SCID mice. To test antibody efficacy on tumorformation, i.p. antibody injections are started on the same day astumor-cell injections. As a control, mice are injected with eitherpurified mouse IgG (ICN) or PBS; or a purified monoclonal antibody thatrecognizes an irrelevant antigen not expressed in human cells. Tumorsizes are determined by caliper measurements, and the tumor volume iscalculated as: Length×Width×Height. Mice with s.c. tumors greater than1.5 cm in diameter are sacrificed.

Orthotopic injections are performed under anesthesia by usingketamine/xylazine. For bladder orthotopic studies, an incision is madethrough the abdomen to expose the bladder, and tumor cells (5×10⁵) mixedwith Matrigel are injected into the bladder wall in a 10-μl volume. Tomonitor tumor growth, mice are palpated and blood is collected on aweekly basis to measure BTA levels. For prostate orthopotic models, anincision is made through the abdominal muscles to expose the bladder andseminal vesicles, which then are delivered through the incision toexpose the dorsal prostate. Tumor cells e.g. LAPC-9 cells (5×10⁵) mixedwith Matrigel are injected into the prostate in a 10-μl volume (YoshidaY et al., Anticancer Res. 1998, 18:327; Ahn et al., Tumour Biol. 2001,22:146). To monitor tumor growth, blood is collected on a weekly basismeasuring PSA levels. Similar procedures are followed for lung andovarian xenograft models. The mice are segregated into groups for theappropriate treatments, with anti-24P4C12 or control mAbs being injectedi.p.

Anti-24P4C12 mAbs Inhibit Growth of 24P4C12-Expressing Xenograft-CancerTumors

The effect of anti-24P4C12 mAbs on tumor formation is tested on thegrowth and progression of bladder, and prostate cancer xenografts usingPC3-24P4C12, Caco-24P4C12, PA-24P4C12, CaLu-24P4C12 or J82-24P4C12orthotopic models. As compared with the s.c. tumor model, the orthotopicmodel, which requires injection of tumor cells directly in the mouseprostate, colon, ovary, lung and bladder, respectively, results in alocal tumor growth, development of metastasis in distal sites,deterioration of mouse health, and subsequent death (Saffran, D., etal., PNAS supra; Fu, X., et al., Int J Cancer, 1992. 52(6): p. 987-90;Kubota, T., J Cell Biochem, 1994. 56(1): p. 4-8). The features make theorthotopic model more representative of human disease progression andallowed us to follow the therapeutic effect of mAbs on clinicallyrelevant end points.

Accordingly, tumor cells are injected into the mouse organs, and 2 dayslater, the mice are segregated into two groups and treated with either:a) 200-500 μg, of anti-24P4C12 Ab, or b) PBS three times per week fortwo to five weeks.

A major advantage of the orthotopic cancer models is the ability tostudy the development of metastases. Formation of metastasis in micebearing established orthotopic tumors is studies by IHC analysis on lungsections using an antibody against a tumor-specific cell-surface proteinsuch as anti-CK20 for bladder cancer, anti-STEAP-1 for prostate cancermodels (Lin S et al, Cancer Detect Prev. 2001; 25:202; Saffran, D., etal., PNAS supra).

Mice bearing established orthotopic tumors are administered 1000 μginjections of either anti-24P4C12 mAb or PBS over a 4-week period. Micein both groups are allowed to establish a high tumor burden, to ensure ahigh frequency of metastasis formation in mouse lungs. Mice then arekilled and their bladders, livers, bone and lungs are analyzed for thepresence of tumor cells by IHC analysis.

These studies demonstrate a broad anti-tumor efficacy of anti-24P4C12antibodies on initiation and progression of prostate and kidney cancerin xenograft mouse models. Anti-24P4C12 antibodies inhibit tumorformation of tumors as well as retarding the growth of alreadyestablished tumors and prolong the survival of treated mice. Moreover,anti-24P4C12 mAbs demonstrate a dramatic inhibitory effect on the spreadof local bladder and prostate tumor to distal sites, even in thepresence of a large tumor burden. Thus, anti-24P4C12 mAbs areefficacious on major clinically relevant end points (tumor growth),prolongation of survival, and health.

Example 39 Therapeutic and Diagnostic use of Anti-24P4C12 Antibodies inHumans

Anti-24P4C12 monoclonal antibodies are safely and effectively used fordiagnostic, prophylactic, prognostic and/or therapeutic purposes inhumans. Western blot and immunohistochemical analysis of cancer tissuesand cancer xenografts with anti-24P4C12 mAb show strong extensivestaining in carcinoma but significantly lower or undetectable levels innormal tissues. Detection of 24P4C12 in carcinoma and in metastaticdisease demonstrates the usefulness of the mAb as a diagnostic and/orprognostic indicator. Anti-24P4C12 antibodies are therefore used indiagnostic applications such as immunohistochemistry of kidney biopsyspecimens to detect cancer from suspect patients.

As determined by flow cytometry, anti-24P4C12 mAb specifically binds tocarcinoma cells. Thus, anti-24P4C12 antibodies are used in diagnosticwhole body imaging applications, such as radioimmunoscintigraphy andradioimmunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res20(2A):925-948 (2000)) for the detection of localized and metastaticcancers that exhibit expression of 24P4C12. Shedding or release of anextracellular domain of 24P4C12 into the extracellular milieu, such asthat seen for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology27:563-568 (1998)), allows diagnostic detection of 24P4C12 byanti-24P4C12 antibodies in serum and/or urine samples from suspectpatients.

Anti-24P4C12 antibodies that specifically bind 24P4C12 are used intherapeutic applications for the treatment of cancers that express24P4C12. Anti-24P4C12 antibodies are used as an unconjugated modalityand as conjugated form in which the antibodies are attached to one ofvarious therapeutic or imaging modalities well known in the art, such asa prodrugs, enzymes or radioisotopes. In preclinical studies,unconjugated and conjugated anti-24P4C12 antibodies are tested forefficacy of tumor prevention and growth inhibition in the SCID mousecancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6,(see, e.g., the Example entitled “24P4C12 Monoclonal Antibody-mediatedInhibition of Bladder and Lung Tumors In Vivo”). Either conjugated andunconjugated anti-24P4C12 antibodies are used as a therapeutic modalityin human clinical trials either alone or in combination with othertreatments as described in following Examples.

Example 40 Human Clinical Trials for the Treatment and Diagnosis ofHuman Carcinomas through use of Human Anti-24P4C12 Antibodies In Vivo

Antibodies are used in accordance with the present invention whichrecognize an epitope on 24P4C12, and are used in the treatment ofcertain tumors such as those listed in Table I. Based upon a number offactors, including 24P4C12 expression levels, tumors such as thoselisted in Table I are presently preferred indications. In connectionwith each of these indications, three clinical approaches aresuccessfully pursued.

I.) Adjunctive therapy: In adjunctive therapy, patients are treated withanti-24P4C12 antibodies in combination with a chemotherapeutic orantineoplastic agent and/or radiation therapy. Primary cancer targets,such as those listed in Table I, are treated under standard protocols bythe addition anti-24P4C12 antibodies to standard first and second linetherapy. Protocol designs address effectiveness as assessed by reductionin tumor mass as well as the ability to reduce usual doses of standardchemotherapy. These dosage reductions allow additional and/or prolongedtherapy by reducing dose-related toxicity of the chemotherapeutic agent.Anti-24P4C12 antibodies are utilized in several adjunctive clinicaltrials in combination with the chemotherapeutic or antineoplastic agentsadriamycin (advanced prostrate carcinoma), cisplatin (advanced head andneck and lung carcinomas), taxol (breast cancer), and doxorubicin(preclinical).

II.) Monotherapy: In connection with the use of the anti-24P4C12antibodies in monotherapy of tumors, the antibodies are administered topatients without a chemotherapeutic or antineoplastic agent. In oneembodiment, monotherapy is conducted clinically in end stage cancerpatients with extensive metastatic disease. Patients show some diseasestabilization. Trials demonstrate an effect in refractory patients withcancerous tumors.

III.) Imaging Agent: Through binding a radionuclide (e.g., iodine oryttrium (I¹³¹, Y⁹⁰) to anti-24P4C12 antibodies, the radiolabeledantibodies are utilized as a diagnostic and/or imaging agent. In such arole, the labeled antibodies localize to both solid tumors, as well as,metastatic lesions of cells expressing 24P4C12. In connection with theuse of the anti-24P4C12 antibodies as imaging agents, the antibodies areused as an adjunct to surgical treatment of solid tumors, as both apre-surgical screen as well as a post-operative follow-up to determinewhat tumor remains and/or returns. In one embodiment, a (¹¹¹In)-24P4C12antibody is used as an imaging agent in a Phase I human clinical trialin patients having a carcinoma that expresses 24P4C12 (by analogy see,e.g., Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991)). Patients arefollowed with standard anterior and posterior gamma camera. The resultsindicate that primary lesions and metastatic lesions are identified

Dose and Route of Administration

As appreciated by those of ordinary skill in the art, dosingconsiderations can be determined through comparison with the analogousproducts that are in the clinic. Thus, anti-24P4C12 antibodies can beadministered with doses in the range of 5 to 400 mg/m², with the lowerdoses used, e.g., in connection with safety studies. The affinity ofanti-24P4C12 antibodies relative to the affinity of a known antibody forits target is one parameter used by those of skill in the art fordetermining analogous dose regimens. Further, anti-24P4C12 antibodiesthat are fully human antibodies, as compared to the chimeric antibody,have slower clearance; accordingly, dosing in patients with such fullyhuman anti-24P4C12 antibodies can be lower, perhaps in the range of 50to 300 mg/m², and still remain efficacious. Dosing in mg/m², as opposedto the conventional measurement of dose in mg/kg, is a measurement basedon surface area and is a convenient dosing measurement that is designedto include patients of all sizes from infants to adults.

Three distinct delivery approaches are useful for delivery ofanti-24P4C12 antibodies. Conventional intravenous delivery is onestandard delivery technique for many tumors. However, in connection withtumors in the peritoneal cavity, such as tumors of the ovaries, biliaryduct, other ducts, and the like, intraperitoneal administration mayprove favorable for obtaining high dose of antibody at the tumor and toalso minimize antibody clearance. In a similar manner, certain solidtumors possess vasculature that is appropriate for regional perfusion.Regional perfusion allows for a high dose of antibody at the site of atumor and minimizes short term clearance of the antibody.

Clinical Development Plan (CDP)

Overview: The CDP follows and develops treatments of anti-24P4C12antibodies in connection with adjunctive therapy, monotherapy, and as animaging agent. Trials initially demonstrate safety and thereafterconfirm efficacy in repeat doses. Trails are open label comparingstandard chemotherapy with standard therapy plus anti-24P4C12antibodies. As will be appreciated, one criteria that can be utilized inconnection with enrollment of patients is 24P4C12 expression levels intheir tumors as determined by biopsy.

As with any protein or antibody infusion-based therapeutic, safetyconcerns are related primarily to (i) cytokine release syndrome, i.e.,hypotension, fever, shaking, chills; (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the antibody therapeutic, or HAHAresponse); and, (iii) toxicity to normal cells that express 24P4C12.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. Anti-24P4C12 antibodies are found to be safe upon humanadministration.

Example 41 Human Clinical Trial Adjunctive Therapy with HumanAnti-24P4C12 Antibody and Chemotherapeutic Agent

A phase I human clinical trial is initiated to assess the safety of sixintravenous doses of a human anti-24P4C12 antibody in connection withthe treatment of a solid tumor, e.g., a cancer of a tissue listed inTable I. In the study, the safety of single doses of anti-24P4C12antibodies when utilized as an adjunctive therapy to an antineoplasticor chemotherapeutic agent as defined herein, such as, withoutlimitation: cisplatin, topotecan, doxorubicin, adriamycin, taxol, or thelike, is assessed. The trial design includes delivery of six singledoses of an anti-24P4C12 antibody with dosage of antibody escalatingfrom approximately about 25 mg/m² to about 275 mg/m² over the course ofthe treatment in accordance with the following schedule:

Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 mAb Dose 25 75 125 175 225 275mg/m² mg/m² mg/m² mg/m² mg/m² mg/m² Chemotherapy + + + + + + (standarddose)

Patients are closely followed for one-week following each administrationof antibody and chemotherapy. In particular, patients are assessed forthe safety concerns mentioned above: (i) cytokine release syndrome,i.e., hypotension, fever, shaking, chills; (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the human antibody therapeutic, or HAHAresponse); and, (iii) toxicity to normal cells that express 24P4C12.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. Patients are also assessed for clinical outcome, andparticularly reduction in tumor mass as evidenced by MRI or otherimaging.

The anti-24P4C12 antibodies are demonstrated to be safe and efficacious,Phase II trials confirm the efficacy and refine optimum dosing.

Example 42 Human Clinical Trial: Monotherapy with Human Anti-24P4C12Antibody

Anti-24P4C12 antibodies are safe in connection with the above-discussedadjunctive trial, a Phase II human clinical trial confirms the efficacyand optimum dosing for monotherapy. Such trial is accomplished, andentails the same safety and outcome analyses, to the above-describedadjunctive trial with the exception being that patients do not receivechemotherapy concurrently with the receipt of doses of anti-24P4C12antibodies.

Example 43 Human Clinical Trial: Diagnostic Imaging with Anti-24P4C12Antibody

Once again, as the adjunctive therapy discussed above is safe within thesafety criteria discussed above, a human clinical trial is conductedconcerning the use of anti-24P4C12 antibodies as a diagnostic imagingagent. The protocol is designed in a substantially similar manner tothose described in the art, such as in Divgi et al. J. Natl. CancerInst. 83:97-104 (1991). The antibodies are found to be both safe andefficacious when used as a diagnostic modality.

Example 44 Homology Comparison of 24P4C12 to Known Sequences

The 24P4C12 protein of FIG. 3 has 710 amino acids with calculatedmolecular weight of 79.3 kDa, and pl of 8.9. Several variants of 24P4C12have been identified, including 4 SNPs (namely v.1, v.3, v.5, v.6) and 3splice variants (namely v.7, v.8 and v.9) (FIGS. 10 and 11). 24P4C12variants v.3, v.5, and v.6 differ from 24P4C12 v.1 by 1 amino acid each,at aa positions 187, 326 and 436, respectively. Variant v.7 carries adeletion of 111 aa long starting at aa 237, while variant v.8 and v.9contain insertions at aa 642 and 378, respectively. The 24P4C12 proteinexhibits homology to a previously cloned human gene, namely NG22 alsoknown as chorine transporter-like protein 4 (gi 14249468). It shows 99%identity and 99% homology to the CTL4 protein over the length of thatprotein (FIG. 4). 24P4C12 is a multi-transmembrane protein, predicted tocarry 10, 11 or 13 transmembrane domains. Bioinformatic analysisindicates that the 24P4C12 protein localizes to the plasma membrane withsome endoplasmic reticulum localization (see Table L). Recent evidenceindicates that the 24P4C12 protein is a 10 transmembrane protein thatlocalizes to the cell surface (O'Regan S et al PNAS 2000, 97:1835).

Choline as an essential component of cell membranes that plays animportant role in cell integrity, growth and survival of normal andtumor cells. Choline accumulates at increased concentration in tumorcells relative to their normal counterparts and as such constitutes atool for the detection of cancer cells by magnetic resonance imaging(Kurhanewicz J et al, J Magn Reson Imaging. 2002.). In addition to itsrole in maintaining membrane integrity, choline mediates signaltransduction event from the membrane to the nucleus (Spiegel S, MilstienS. J Membr Biol. 1995, 146:225). Choline metabolites includesphingosylphosphorylcholine and Iysophosphatidylcholine, both of whichactivate G-protein coupled receptors (Xu F et al Biochim Biophys Acta2002, 1582:81). In addition, choline results in the activation of kinasepathways including Raf-1 (Lee M, Han S S, Cell Signal 2002, 14:373.).Choline also plays a role in regulating DNA methylation and regulationof gene expression. For example, choline methanolites regulate theexpression of cytokines and chemokines essential for tumor growth(Schwartz B M et al., Gynecol Oncol. 2001, 81:291; Denda A et al.,Carcinogenesis. 2002, 23:245). Due to its effect on cell signaling andgene expression, choline controls cell growth and survival(Holmes-McNary M Q et al, J Biol Chem. 2001, 276: 41197; Albright etal., FASEB 1996, 10:510). Choline deficiency results in cell death,apoptosis and transformation, while accumulation of choline isassociated with tumor growth (Zeisel S et al, Carcinogenesis 1997,18:731). Accordingly, when 24P4C12 functions as a regulator of tumorformation, cell proliferation, invasion or cell signaling, 24P4C12 isused for therapeutic, diagnostic, prognostic and/or preventativepurposes.

Example 45 Identification and Confirmation of Potential SignalTransduction Pathways

Many mammalian proteins have been reported to interact with signalingmolecules and to participate in regulating signaling pathways. (JNeurochem. 2001; 76:217-223). In particular, choline have been reportedto activate MAK cascades as well as G proteins, and been associated withthe DAG and ceramide and sphingophosphorylcholine signaling pathway(Cummings et al, above). In addition, choline transmit its signals byregulating choline-kinase and phospholipase activity, resulting inenhance tumorigenic effect (Ramirez et al, Oncogene. 2002, 21:4317;Lucas et al., Oncogene. 2001, 20:1110; Chung T et al, Cell Signal. 2000,12:279).

Using immunoprecipitation and Western blotting techniques, proteins areidentified that associate with 24P4C12 and mediate signaling events.Several pathways known to play a role in cancer biology can be regulatedby 24P4C12, including phospholipid pathways such as PI3K, AKT, etc,adhesion and migration pathways, including FAK, Rho, Rac-1, etc, as wellas mitogenic/survival cascades such as ERK, p38, etc (Cell GrowthDiffer. 2000, 11:279; J Biol Chem. 1999, 274:801; Oncogene. 2000,19:3003; J. Cell Biol. 1997, 138:913). Using Western blotting and othertechniques, the ability of 24P4C12 to regulate these pathways isconfirmed. Cells expressing or lacking 24P4C12 are either left untreatedor stimulated with cytokines, androgen and anti-integrin antibodies.Cell lysates are analyzed using anti-phospho-specific antibodies (CellSignaling, Santa Cruz Biotechnology) in order to detect phosphorylationand regulation of ERK, p38, AKT, PI3K, PLC and other signalingmolecules.

To confirm that 24P4C12 directly or indirectly activates known signaltransduction pathways in cells, luciferase (luc) based transcriptionalreporter assays are carried out in cells expressing individual genes.These transcriptional reporters contain consensus-binding sites forknown transcription factors that lie downstream of well-characterizedsignal transduction pathways. The reporters and examples of theseassociated transcription factors, signal transduction pathways, andactivation stimuli are listed below.

1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress

2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation

3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress

4. ARE-luc, androgen receptor; steroids/MAPK;growth/differentiation/apoptosis

5. p53-luc, p53; SAPK; growth/differentiation/apoptosis

6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

7. TCF-luc, TCF/Lef;

-catenin, Adhesion/invasion

Gene-mediated effects can be assayed in cells showing mRNA expression.Luciferase reporter plasmids can be introduced by lipid-mediatedtransfection (TFX-50, Promega). Luciferase activity, an indicator ofrelative transcriptional activity, is measured by incubation of cellextracts with luciferin substrate and luminescence of the reaction ismonitored in a luminometer.

Signaling pathways activated by 24P4C12 are mapped and used for theidentification and validation of therapeutic targets. When 24P4C12 isinvolved in cell signaling, it is used as target for diagnostic,prognostic, preventative and/or therapeutic purposes.

Example 46 24P4C12 Functions as a Choline Transporter

Sequence and homology analysis of 24P4C12 indicate that 24P4C12 carriesa transport domain and that 24P4C12 functions as a choline transporter.In order to confirm that 24P4C12 transports choline, primary and tumorcells, including prostate, colon, bladder and lung lines, are grown inthe presence and absence of ³H-choline. Radioactive choline uptake ismeasured by counting incorporated counts per minutes (cpm). Parental24P4C12 negative cells are compared to 24P4C12-expressing cells usingthis and similar assays. Similarly, parental and 24P4C12-expressingcells can be compared for choline content using NMR spectroscopy. Theseassay systems can be used to identify small molecules and antibodiesthat interfere with choline uptake and/or with the function of 24P4C12.

Thus, compounds and small molecules designed to inhibit 24P4C12 functionand downstream signaling events are used for therapeutic diagnostic,prognostic and/or preventative purposes.

Example 47 Regulation of Transcription

The cell surface localization of 24P4C12 and its ability to regulate DNAmethylation indicate that it is effectively used as a modulator of thetranscriptional regulation of eukaryotic genes. Regulation of geneexpression is confirmed, e.g., by studying gene expression in cellsexpressing or lacking 24P4C12. For this purpose, two types ofexperiments are performed.

In the first set of experiments, RNA from parental and24P4C12-expressing cells are extracted and hybridized to commerciallyavailable gene arrays (Clontech) (Smid-Koopman E et al. Br J Cancer.2000. 83:246). Resting cells as well as cells treated with FBS,pheromones, or growth factors are compared. Differentially expressedgenes are identified in accordance with procedures known in the art. Thedifferentially expressed genes are then mapped to biological pathways(Chen K et al. Thyroid. 2001. 11:41.).

In the second set of experiments, specific transcriptional pathwayactivation is evaluated using commercially available (Stratagene)luciferase reporter constructs including: NFkB-luc, SRE-luc, ELK1-luc,ARE-luc, p53-luc, and CRE-luc. These transcriptional reporters containconsensus binding sites for known transcription factors that liedownstream of well-characterized signal transduction pathways, andrepresent a good tool to ascertain pathway activation and screen forpositive and negative modulators of pathway activation.

Thus, 24P4C12 plays a role in gene regulation, and it is used as atarget for diagnostic, prognostic, preventative and/or therapeuticpurposes.

Example 48 Involvement in Tumor Progression

The 24P4C12 gene can contribute to the growth of cancer cells. The roleof 24P4C12 in tumor growth is confirmed in a variety of primary andtransfected cell lines including prostate, and bladder cell lines, aswell as NIH 3T3 cells engineered to stably express 24P4C12. Parentalcells lacking 24P4C12 and cells expressing 24P4C12 are evaluated forcell growth using a well-documented proliferation assay (Fraser S P, etal., Prostate 2000; 44:61, Johnson D E, Ochieng J, Evans S L. AnticancerDrugs. 1996, 7:288). Such a study was performed on prostate cancer cellsand the results are shown in FIG. 28. The growth of parental PC3 andPC3-24P4C12 cells was compared in low (0.1%) and 10% FBS. Expression of24P4C12 imparted a growth advantage to PC3 cells grown in 10% FBS.Similarly, expression of 24P4C12 in NIH-3T3 cells enhances theproliferation of these cells relative to control 3T3-neo cells. Theeffect of 24P4C12 can also be observed on cell cycle progression.Control and 24P4C12-expressing cells are grown in low serum overnight,and treated with 10% FBS for 48 and 72 hrs. Cells are analyzed for BrdUand propidium iodide incorporation by FACS analysis.

To confirm the role of 24P4C12 in the transformation process, its effectin colony forming assays is investigated. Parental NIH-3T3 cells lacking24P4C12 are compared to NIH-3T3 cells expressing 24P4C12, using a softagar assay under stringent and more permissive conditions (Song Z. etal. Cancer Res. 2000; 60:6730).

To confirm the role of 24P4C12 in invasion and metastasis of cancercells, a well-established assay is used. A non-limiting example is theuse of an assay which provides a basement membrane or an analog thereofused to detect whether cells are invasive (e.g., a Transwell InsertSystem assay (Becton Dickinson) (Cancer Res. 1999; 59:6010)). Controlcells, including prostate, and bladder cell lines lacking 24P4C12 arecompared to cells expressing 24P4C12. Cells are loaded with thefluorescent dye, calcein, and plated in the top well of a supportstructure coated with a basement membrane analog (e.g. the Transwellinsert) and used in the assay. Invasion is determined by fluorescence ofcells in the lower chamber relative to the fluorescence of the entirecell population.

24P4C12 can also play a role in cell cycle and apoptosis. Parental cellsand cells expressing 24P4C12 are compared for differences in cell cycleregulation using a well-established BrdU assay (Abdel-Malek Z A. J CellPhysiol. 1988, 136:247). In short, cells are grown under both optimal(full serum) and limiting (low serum) conditions are labeled with BrdUand stained with anti-BrdU Ab and propidium iodide. Cells are analyzedfor entry into the G1, S, and G2M phases of the cell cycle.Alternatively, the effect of stress on apoptosis is evaluated in controlparental cells and cells expressing 24P4C12, including normal and tumorprostate, colon and lung cells. Engineered and parental cells aretreated with various chemotherapeutic agents, such as etoposide,flutamide, etc, and protein synthesis inhibitors, such as cycloheximide.Cells are stained with annexin V-FITC and cell death is measured by FACSanalysis. The modulation of cell death by 24P4C12 can play a criticalrole in regulating tumor progression and tumor load.

When 24P4C12 plays a role in cell growth, transformation, invasion orapoptosis, it is used as a target for diagnostic, prognostic,preventative and/or therapeutic purposes.

Example 49 Involvement in Angiogenesis

Angiogenesis or new capillary blood vessel formation is necessary fortumor growth (Hanahan D, Folkman J. Cell. 1996, 86:353; Folkman J.Endocrinology. 1998 139:441). Based on the effect of phsophodieseteraseinhibitors on endothelial cells, 24P4C12 plays a role in angiogenesis(DeFouw L et al, Microvasc Res 2001, 62:263). Several assays have beendeveloped to measure angiogenesis in vitro and in vivo, such as thetissue culture assays endothelial cell tube formation and endothelialcell proliferation. Using these assays as well as in vitroneo-vascularization, the role of 24P4C12 in angiogenesis, enhancement orinhibition, is confirmed.

For example, endothelial cells engineered to express 24P4C12 areevaluated using tube formation and proliferation assays. The effect of24P4C12 is also confirmed in animal models in vivo. For example, cellseither expressing or lacking 24P4C12 are implanted subcutaneously inimmunocompromised mice. Endothelial cell migration and angiogenesis areevaluated 5-15 days later using immunohistochemistry techniques. 24P4C12affects angiogenesis and it is used as a target for diagnostic,prognostic, preventative and/or therapeutic purposes.

Example 50 Involvement in Adhesion

Cell adhesion plays a critical role in tissue colonization andmetastasis. The presence of leucine rich and cysteine rich motifs in24P4C12 is indicative of its role in cell adhesion. To confirm that24P4C12 plays a role in cell adhesion, control cells lacking 24P4C12 arecompared to cells expressing 24P4C12, using techniques previouslydescribed (see, e.g., Haier et al, Br. J. Cancer. 1999, 80:1867; Lehrand Pienta, J. Natl. Cancer Inst. 1998, 90:11). Briefly, in oneembodiment, cells labeled with a fluorescent indicator, such as calcein,are incubated on tissue culture wells coated with media alone or withmatrix proteins. Adherent cells are detected by fluorimetric analysisand percent adhesion is calculated. This experimental system can be usedto identify proteins, antibodies and/or small molecules that modulatecell adhesion to extracellular matrix and cell-cell interaction. Sincecell adhesion plays a critical role in tumor growth, progression, and,colonization, the gene involved in this process can serves as adiagnostic, preventative and therapeutic modality.

Example 51 Detection of 24P4C12 Protein in Cancer Patient Specimens

To determine the expression of 24P4C12 protein, specimens were obtainedfrom various cancer patients and stained using an affinity purifiedpolyclonal rabbit antibody raised against the peptide encoding aminoacids 1-14 of 24P4C12 variant 1 and conjugated to KLH (See, Example 10:Generation of 24P4C12 Polyclonal Antibodies.) This antiserum exhibited ahigh titer to the peptide (>10,000) and recognized 24P4C12 intransfected 293T cells by Western blot and flow cytometry (FIG. 24) andin stable recombinant PC3 cells by Western blot and immunohistochemistry(FIG. 25). Formalin fixed, paraffin embedded tissues were cut into 4micron sections and mounted on glass slides. The sections were dewaxed,rehydrated and treated with antigen retrieval solution (0.1 M Tris,pH10) at high temperature. Sections were then incubated in polyclonalrabbit anti-24P4C12 antibody for 3 hours. The slides were washed threetimes in buffer and further incubated with DAKO EnVision+™peroxidase-conjugated goat anti-rabbit immunoglobulin secondary antibody(DAKO Corporation, Carpenteria, Calif.) for 1 hour. The sections werethen washed in buffer, developed using the DAB kit (SIGMA Chemicals),counterstained using hematoxylin, and analyzed by bright fieldmicroscopy. The results showed expression of 24P4C12 in cancer patients'tissue (FIGS. 29 and 30). Tissue from prostate cancer patients showedexpression of 24P4C12 in the tumor cells and in the prostate epitheliumof tissue normal adjacent to tumor (FIG. 29). Generally, expression of24P4C12 was high in all prostate tumors and was expressed mainly aroundthe cell membrane indicating that 24P4C12 is membrane associated inprostate tissues. All of the prostate samples tested were positive for24P4C12. Other tumors that were positive for 24P4C12 included colonadenocarcinoma, breast ductal carcinoma, pancreatic adenocarcinoma, lungadenocarcinoma, bladder transitional cell carcinoma and renal clear cellcarcinoma (FIG. 30). Normal tissues investigated for expression of24P4C12 included heart, skeletal muscle, liver, brain, spinal cord,skin, adrenal, lymph node, spleen, salivary gland, small intestine andplacenta. None demonstrated any expression of 24P4C12 byimmunohistochemistry. Normal adjacent to tumor tissues were also studiedto determine the presence of 24P4C12 protein by immunohistochemistry.These included breast, lung, colon, ileum, bladder, kidney and pancreas.In some of the tissues from these organs there was weak expression of24P4C12. This expression may relate to the fact that the samples werenot truly normal and may indicate a precancerous change. The ability toidentify malignancy in tissue that has not undergone obviousmorphological changes is an important diagnostic modality for cancerousand precancerous conditions.

These results indicate that 24P4C12 is a target for diagnostic,prophylactic, prognostic and therapeutic applications in cancer.

Throughout this application, various website data content, publications,patent applications and patents are referenced. (Websites are referencedby their Uniform Resource Locator, or URL, addresses on the World WideWeb.) The disclosures of each of these references are herebyincorporated by reference herein in their entireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

Tables:

TABLE I Tissues that Express 24P4C12: a. Malignant Tissues ProstateBladder Kidney Lung Colon Ovary Breast Uterus Stomach

TABLE II Amino Acid Abbreviations SINGLE LETTER THREE LETTER FULL NAME FPhe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cyscysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamineR Arg arginme I Ile isoleucine M Met methionine T Thr threonine N Asnasparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid EGlu glutamic acid G Gly glycine

TABLE III Amino Acid Substitution Matrix Adapted from the GCG Software9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix).The higher the value, the more likely a substitution is found inrelated, natural proteins. (See world wide web URLikp.unibe.ch/manual/blosum62.html) A C D E F G H I K L M N P Q R S T V WY . 4 0 −2 −1 −2 0 −2 −1 −1 −1 −1 −2 −1 −1 −1 1 0 0 −3 −2 A 9 −3 −4 −2−3 −3 −1 −3 −1 −1 −3 −3 −3 −3 −1 −1 −1 −2 −2 C 6 2 −3 −1 −1 −3 −1 −4 −31 −1 0 −2 0 −1 −3 −4 −3 D 5 −3 −2 0 −3 1 −3 −2 0 −1 2 0 0 −1 −2 −3 −2 E6 −3 −1 0 −3 0 0 −3 −4 −3 −3 −2 −2 −1 1 3 F 6 −2 −4 −2 −4 −3 0 −2 −2 −20 −2 −3 −2 −3 G 8 −3 −1 −3 −2 1 −2 0 0 −1 −2 −3 −2 2 H 4 −3 2 1 −3 −3 −3−3 −2 −1 3 −3 −1 I 5 −2 −1 0 −1 1 2 0 −1 −2 −3 −2 K 4 2 −3 −3 −2 −2 −2−1 1 −2 −1 L 5 −2 −2 0 −1 −1 −1 1 −1 −1 M 6 −2 0 0 1 0 −3 −4 −2 N 7 −1−2 −1 −1 −2 −4 −3 P 5 1 0 −1 −2 −2 −1 Q 5 −1 −1 −3 −3 −2 R 4 1 −2 −3 −2S 5 0 −2 −2 T 4 −3 −1 V 11 2 W 7 Y

Table IV: HLA Class I/II Motifs/Supermotifs

TABLE IV (A) HLA Class I Supermotifs/Motifs POSITION POSITION POSITION CTerminus (Primary 2 (Primary Anchor) 3 (Primary Anchor) Anchor)SUPERMOTIF A1 TI LVMS FWY A2 LIVM ATQ IV MATL A3 VSMA TLI RK A24 YFWIVLMT FI YWLM B7 P VILF MWYA B27 RHK FYL WMIVA B44 E D FWYLIMVA B58 ATSFWY LIVMA B62 QL IVMP FWY MIVLA MOTIFS A1 TSM Y A1 DE AS Y A2.1 LM VQIATV LIMAT A3 LMVISATF CGD KYR HFA A11 VTMLISAGN CDF K RYH A24 YF WM FLIWA*3101 MVT ALIS R K A*3301 MVALF IST RK A*6801 AVT MSLI RK B*0702 P LMFWYAIV B*3501 P LMFWY IVA B51 P LIVF WYAM B*5301 P IMFWY ALV B*5401 PATIV LMFWY Bolded residues are preferred, italicized residues are lesspreferred: A peptide is considered motif-bearing if it has primaryanchors at each primary anchor position for a motif or supermotif asspecified in the above table.

TABLE IV (B) HLA Class II Supermotif 1 6 9 W, F, Y, V, .I, L A, V, I, L,P, C, S, T A, V, I, L, C, S, T, M, Y

TABLE IV (C) HLA Class II Motifs MOTIFS 1° anchor 1 2 3 4 5 1° anchor 67 8 9 DR4 preferred FMYLIVW M T I VSTCPALIM MH MH deleterious W R WDEDR1 preferred MFLIVWY PAMQ CWD VMATSPLIC M AVM deleterious C CH FD GDE DDR7 preferred MFLIVWY M W A IVMSACTPL M IV deleterious C G GRD N G DR3MOTIFS 1° anchor 1 2 3 1° anchor 4 5 1° anchor 6 Motif a preferredLIVMFY D Motif b preferred LIVMFAY DNQEST KRH DR Supermotif MFLIVWYVMSTACPLI Italicized residues indicate less preferred or “tolerated”residues

TABLE IV (D) HLA Class I Supermotifs SUPER- POSITION: MOTIFS 1 2 3 4 5 67 8 C-terminus A1 1° Anchor 1° Anchor TILVMS FWY A2 1° Anchor 1° AnchorLIVMATQ LIVMAT A3 Preferred 1° Anchor YFW YFW YFW P 1° Anchordeleterious DE (3/5); VSMATLI (4/5) (3/5) (4/5) (4/5) RK P (5/5) DE(4/5) A24 1° Anchor 1° Anchor YFWIVLMT FIYWLM B7 Preferred FWY (5/5) 1°Anchor FWY FWY 1° Anchor LIVM (3/5) P (4/5) (3/5) VILFMWYA deleteriousDE (3/5); DE G QN DE P(5/5); (3/5) (4/5) (4/5) (4/5) G(4/5); A(3/5);QN(3/5) B27 1° Anchor 1° Anchor RHK FYLWMIVA B44 1° Anchor 1° Anchor EDFWYLIMVA B58 1° Anchor 1° Anchor ATS FWYLIVMA B62 1° Anchor 1° AnchorQLIVMP FWYMIVLA Italicized residues indicate less preferred or“tolerated” residues

TABLE IV (E) HLA Class I Motifs POSITION 1 2 3 4 5 A1 preferred GFYW 1°Anchor DEA YFW 9-mer STM deleterious DE RHKLIVMP A G A1 preferred GRHKASTCLIVM 1° Anchor GSTC 9-mer DEAS deleterious A RHKDEPYFW DE PQN A1preferred YFW 1° Anchor DEAQN A YFWQN 10-mer STM deleterious GP RHKGLIVMDE RHK A1 preferred YFW STCLIVM 1° Anchor A YFW 10-mer DEAS deleteriousRHK RHKDEPYFW P A2.1 preferred YFW 1°Anchor YFW STC YFW 9-mer LMIVQATdeleterious DEP DERKH POSITION 9 or 6 7 8 C-terminus C-terminus A1preferred P DEQN YFW 1° Anchor 9-mer Y deleterious A A1 preferred ASTCLIVM DE 1° Anchor 9-mer Y deleterious RHK PG GP A1 preferred PASTC GDE P1° Anchor 10-mer Y deleterious QNA RHKYFW RHK A A1 preferred PG G YFW 1°Anchor 10-mer Y deleterious G PRHK QN A2.1 preferred A P 1° Anchor 9-merVLIMAT deleterious RKH DERKH POSITION: 1 2 3 4 5 A2.1 preferred AYFW 1°Anchor LVIM G 10-mer LMIVQAT deleterious DEP DE RKHA P A3 preferred RHK1° Anchor YFW PRHKYFW A LMVISATFCGD deleterious DEP DE A11 preferred A1° Anchor YFW YFW A VTLMISAGNCDF deleterious DEP A24 preferred YFWRHK 1°Anchor STC 9-mer YFWM deleterious DEG DE G QNP A24 Preferred 1° Anchor PYFWP 10-mer YFWM Deleterious GDE QN RHK A3101 Preferred RHK 1° AnchorYFW P MVTALIS Deleterious DEP DE ADE A3301 Preferred 1° Anchor YFWMVALFIST Deleterious GP DE A6801 Preferred YFWSTC 1° Anchor YFWLIVMAVTMSLI deleterious GP DEG RHK B0702 Preferred RHKFWY 1° Anchor RHK RHKP deleterious DEQNP DEP DE DE POSITION: 6 7 8 9 C-terminus A2.1preferred G FYWL 1° Anchor 10-mer VIM VLIMAT deleterious RKH DERKHRKH A3preferred YFW P 1° Anchor KYRHFA deleterious A11 preferred YFW YFW P 1°Anchor KRYH deleterious A G A24 preferred YFW YFW 1° Anchor 9-mer FLIWdeleterious DERHK G AQN A24 Preferred P 1° Anchor 10-mer FLIWDeleterious DE A QN DEA A3101 Preferred YFW YFW AP 1° Anchor RKDeleterious DE DE DE A3301 Preferred AYFW 1° Anchor RK Deleterious A6801Preferred YFW P 1° Anchor RK deleterious A B0702 Preferred RHK RHK PA 1°Anchor LMFWYAIV POSITION 1 2 3 4 5 A1 preferred GFYW 1° Anchor DEA YFW9-mer STM deleterious DE RHKLIVMP A G A1 preferred GRHK ASTCLIVM 1°Anchor GSTC 9-mer DEAS deleterious A RHKDEPYFW DE PQN B3501 PreferredFWYLIVM 1° Anchor FWY P deleterious AGP G B51 Preferred LIVMFWY 1°Anchor FWY STC FWY P deleterious AGPDER DE HKSTC B5301 preferred LIVMFWY1° Anchor FWY STC FWY P deleterious AGPQN B5401 preferred FWY 1° AnchorFWYLIVM LIVM P deleterious GPQNDE GDESTC RHKDE POSITION 9 or 6 7 8C-terminus C-terminus A1 preferred P DEQN YFW 1° Anchor 9-mer Ydeleterious A A1 preferred ASTC LIVM DE 1° Anchor 9-mer Y deleteriousRHK PG GP B3501 Preferred FWY 1° Anchor LMFWYIVA deleterious G B51Preferred G FWY 1° Anchor LIVFWYAM deleterious G DEQN GDE B5301preferred LIVMFWY FWY 1° Anchor IMFWYALV deleterious G RHKQN DE B5401preferred ALIVM FWYA 1° Anchor P ATIVLMFWY deleterious DE QNDGE DE

TABLE IV (F) Summary of HLA-supertypes Overall phenotypic frequencies ofHLA- supertypes in different ethnic populations Specificity Phenotypicfrequency Supertype Position 2 C-Terminus Caucasian N.A. Black JapaneseChinese Hispanic Average B7 P AILMVFWY 43.2 55.1 57.1 43.0 49.3 49.5 A3AILMVST RK 37.5 42.1 45.8 52.7 43.1 44.2 A2 AILMVT AILMVT 45.8 39.0 42.445.9 43.0 42.2 A24 YF (WIVLMT) FI (YWLM) 23.9 38.9 58.6 40.1 38.3 40.0B44 E (D) FWYLIMVA 43.0 21.2 42.9 39.1 39.0 37.0 A1 TI (LVMS) FWY 47.116.1 21.8 14.7 26.3 25.2 B27 RHK FYL (WMI) 28.4 26.1 13.3 13.9 35.3 23.4B62 QL (IVMP) FWY (MIV) 12.6 4.8 36.5 25.4 11.1 18.1 B58 ATS FWY (LIV)10.0 25.1 1.6 9.0 5.9 10.3

TABLE IV (G) Calculated population coverage afforded by differentHLA-supertype combinations Phenotypic frequency HLA-supertypes CaucasianN.A Blacks Japanese Chinese Hispanic Average A2, A3 and B7 83.0 86.187.5 88.4 86.3 86.2 A2, A3, B7, A24, 99.5 98.1 100.0 99.5 99.4 99.3 B44and A1 A2, A3, 99.9 99.6 100.0 99.8 99.9 99.8 B7, A24, B44, A1, B27,B62, and B 58 Motifs indicate the residues defining supertypespecificites. The motifs incorporate residues determined on the basis ofpublished data to be recognized by multiple alleles within thesupertype. Residues within brackets are additional residues alsopredicted to be tolerated by multiple alleles within the supertype.

TABLE V Frequently Occurring Motifs avrg. % Name identity DescriptionPotential Function zf-C2H2 34% Zinc finger, C2H2 type Nucleicacid-binding protein functions as transcription factor, nuclear locationprobable cytochrome_b_N 68% Cytochrome b(N- membrane bound oxidase,generate terminal)/b6/petB superoxide Ig 19% Immunoglobulin domaindomains are one hundred amino acids long and include a conservedintradomain disulfide bond. WD40 18% WD domain, G-beta repeat tandemrepeats of about 40 residues, each containing a Trp-Asp motif. Functionin signal transduction and protein interaction PDZ 23% PDZ domain mayfunction in targeting signaling molecules to sub-membranous sites LRR28% Leucine Rich Repeat short sequence motifs involved inprotein-protein interactions Pkinase 23% Protein kinase domain conservedcatalytic core common to both serine/threonine and tyrosine proteinkinases containing an ATP binding site and a catalytic site PH 16% PHdomain pleckstrin homology involved in intracellular signaling or asconstituents of the cytoskeleton EGF 34% EGF-like domain 30-40amino-acid long found in the extracellular domain of membrane- boundproteins or in secreted proteins Rvt 49% Reverse transcriptase(RNA-dependent DNA polymerase) Ank 25% Ank repeat Cytoplasmic protein,associates integral membrane proteins to the cytoskeleton Oxidored_q132% NADH- membrane associated. Involved in Ubiquinone/plastoquinoneproton translocation across the (complex I), various chains membraneEfhand 24% EF hand calcium-binding domain, consists of a12 residue loopflanked on both sides by a 12 residue alpha-helical domain Rvp 79%Retroviral aspartyl Aspartyl or acid proteases, centered on protease acatalytic aspartyl residue Collagen 42% Collagen triple helix repeatextracellular structural proteins involved (20 copies) in formation ofconnective tissue. The sequence consists of the G-X-Y and thepolypeptide chains forms a triple helix. Fn3 20% Fibronectin type IIIdomain Located in the extracellular ligand- binding region of receptorsand is about 200 amino acid residues long with two pairs of cysteinesinvolved in disulfide bonds 7tm_1 19% 7 transmembrane receptor sevenhydrophobic transmembrane (rhodopsin family) regions, with theN-terminus located extracellularly while the C-terminus is cytoplasmic.Signal through G proteins

TABLE VI Motifs and Post-translational Modifications of 24P4C12N-glycosylation site  29-32 NRSC (SEQ ID NO: 48)  69-72 NSTG (SEQ ID NO:49) 155-158 NMTV (SEQ ID NO: 50) 197-200 NDTT (SEQ ID NO: 51) 298-301NLSA (SEQ ID NO: 52) 393-396 NISS (SEQ ID NO: 53) 405-408 NTSC (SEQ IDNO: 54) 416-419 NSSC (SEQ ID NO: 55) 678-681 NGSL (SEQ ID NO: 56)Protein kinase C phosphorylation site  22-24 SfR 218-220 SvK 430-432 SsK494-496 TIR 573-575 SaK 619-621 SgR Casein kinase II phosphorylationsite  31-34 SCTD (SEQ ID NO: 57) 102-105 SVAE (SEQ ID NO: 58) 119-122SCPE (SEQ ID NO: 59) 135-138 TVGE (SEQ ID NO: 60) 304-307 SVQE (SEQ IDNO: 61) Tyrosine kinase phosphorylation site   6-13 RDEDDEAY (SEQ ID NO:62) N-myristoylation site  72-77 GAYCGM (SEQ ID NO: 63)  76-81 GMGENK(SEQ ID NO: 64) 151-156 GVPWNM (SEQ ID NO: 65) 207-212 GLIDSL (SEQ IDNO: 66) 272-277 GIYYCW (SEQ ID NO: 67) 287-292 GASISQ (SEQ ID NO: 68)349-354 GQMMST (SEQ ID NO: 69) 449-454 GLFWTL (SEQ ID NO: 70) 467-472GAFASF (SEQ ID NO: 71) Amidation site 695-698 IGKK (SEQ ID NO: 72)Leucine zipper pattern 245-266 LFILLLRLVAGPLVLVLILGVL (SEQ ID NO: 73)Cysteine-rich region 536-547 CIMCCFKCCLWC (SEQ ID NO: 74)

TABLE VII Search Peptides Variant 1, 9-mers, 10-mers, 15-mers (SEQ IDNO: 75) MGGKQRDEDD EAYGKPVKYD PSFRGPIKNR SCTDVICCVL FLLFILGYIVVGIVAWLYGD PRQVLYPRNS TGAYCGMGEN KDKPYLLYFN IFSCILSSNI ISVAENGLQCPTPQVCVSSC PEDPWTVGKN EFSQTVGEVF YTKNRNFCLP GVPWNMTVIT SLQQELCPSFLLPSAPALGR CFPWTNVTPP ALPGITNDTT IQQGISGLID SLNARDISVK IFEDFAQSWYWILVALGVAL VLSLLFILLL RLVAGPLVLV LILGVLGVLA YGIYYCWEEY RVLRDKGASISQLGFTTNLS AYQSVQETWL AALIVLAVLE AILLLMLIFL RQRIRIAIAL LKEASKAVGQMMSTMFYPLV TFVLLLICIA YWAMTALYLA TSGQPQYVLW ASNISSPGCE KVPINTSCNPTAHLVNSSCP GLMCVFQGYS SKGLIQRSVF NLQIYGVLGL FWTLNWVLAL GQCVLAGAFASFYWAFHKPQ DIPTFPLISA FIRTLRYHTG SLAFGALILT LVQIARVILE YIDHKLRGVQNPVARCIMCC FKCCLWCLEK FIKFLNRNAY IMIAIYGKNF CVSAKNAFML LMRNIVRVVVLDKVTDLLLF FGKLLVVGGV GVLSFFFFSG RIPGLGKDFK SPHLNYYWLP IMTSILGAYVIASGFFSVFG MCVDTLFLCF LEDLERNNGS LDRPYYMSKS LLKILGKKNE APPDNKKRKKVariant 3: 9-mers (SEQ ID NO: 76) GRCFPWTNITPPALPGI 10-mers (SEQ ID NO:77) LGRCFPWTNITPPALPGIT 15-mers (SEQ ID NO: 78)PSAPALGRCFPWTNITPPALPGITNDTTI Variant 5: 9-mers (SEQ ID NO: 79)VLEAILLLVLIFLRQRI 10-mers (SEQ ID NO: 80) AVLEAILLLVLIFLRQRIR 15-mers(SEQ ID NO: 81) ALIVLAVLEAILLLVLIFLRQRIRIAIAL Variant 6: 9-mers (SEQ IDNO: 82) GYSSKGLIPRSVFNLQI 10-mers (SEQ ID NO: 83) QGYSSKGLIPRSVFNLQIY15-mers (SEQ ID NO: 84) LMCVFQGYSSKGLIPRSVFNLQIYGVLGL Variant 7 9-mers(SEQ ID NO: 85) SWYWILVAVGQMMSTM 10-mers (SEQ ID NO: 86)QSWYWILVAVGQMMSTMF 15-mers (SEQ ID NO: 87) FEDFAQSWYWILVAVGQMMSTMFYPLVTVariant 8 9-mers (SEQ ID NO: 88) NYYWLPIMRNPITPTGHVFQTSILGAYV 10-mers(SEQ ID NO: 89) LNYYWLPIMRNPITPTGHVFQTSILGAYVI 15-mers (SEQ ID NO: 90)FKSPHLNYYWLPIMRNPITPTGHVFQTSILGAYVIASGFF Variant 9 9-mers (SEQ ID NO:91) YWAMTALYPLPTQPATLGYVLWASNI 10-mers (SEQ ID NO: 92)AYWAMTALYPLPTQPATLGYVLWASNIS 15-mers (SEQ ID NO: 93)LLICIAYWAMTALYPLPTQPATLGYVLWASNISSPGCE

Tables VII-XXI:

TABLE VIII V1-HLA-A1-9mers- 24P4C12 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 58 YGDPRQVLY 125.000 662 CVDTLFLCF25.000 77 MGENKDKPY 11.250 594 VTDLLLFFG 6.250 698 KNEAPPDNK 4.500 318VLEAILLLM 4.500 363 VLLLICIAY 2.500 489 SAFIRTLRY 2.500 267 GVLAYGIYY2.500 689 KSLLKILGK 1.500 470 ASFYWAFHK 1.500 222 FEDFAQSWY 1.250 32CTDVICCVL 1.250 5 QRDEDDEAY 1.250 121 PEDPWTVGK 1.000 379 LATSGQPQY1.000 700 EAPPDNKKR 1.000 558 NAYIMIAIY 1.000 542 KCCLWCLEK 1.000 7DEDDEAYGK 1.000 11 EAYGKPVKY 1.000 670 FLEDLERNN 0.900 276 CWEEYRVLR0.900 518 ILEYIDHKL 0.900 417 SSCPGLMCV 0.750 437 RSVFNLQIY 0.750 80NKDKPYLLY 0.625 263 LGVLGVLAY 0.625 546 WCLEKFIKF 0.500 243 SLLFILLLR0.500 238 VALVLSLLF 0.500 579 MLLMRNIVR 0.500 465 LAGAFASFY 0.500 421GLMCVFQGY 0.500 508 ILTLVQIAR 0.500 593 KVTDLLLFF 0.500 321 AILLLMLIF0.500 36 ICCVLFLLF 0.500 50 VVGIVAWLY 0.500 186 NVTPPALPG 0.500 609GVGVLSFFF 0.500 287 GASISQLGF 0.500 187 VTPPALPGI 0.500 668 LCFLEDLER0.500 323 LLLMLIFLR 0.500 272 GIYYCWEEY 0.500 521 YIDHKLRGV 0.500 253VAGPLVLVL 0.500 398 GCEKVPINT 0.450 560 YIMIAIYGK 0.400 338 IALLKEASK0.400 135 TVGEVFYTK 0.400 349 GQMMSTMFY 0.375 118 SSCPEDPWT 0.300 305VQETWLAAL 0.270 629 FKSPHLNYY 0.250 214 ARDISVKIF 0.250 702 PPDNKKRKK0.250 641 IMTSILGAY 0.250 678 NGSLDRPYY 0.250 513 QIARVILEY 0.250 483PTFPLISAF 0.250 120 CPEDPWTVG 0.225 129 KNEFSQTVG 0.225 136 VGEVFYTKN0.225 170 FLLPSAPAL 0.200 147 FCLPGVPWN 0.200 393 NISSPGCEK 0.200 464VLAGAFASF 0.200 517 VILEYIDHK 0.200 424 CVFQGYSSK 0.200 394 ISSPGCEKV0.150 133 SQTVGEVFY 0.150 613 LSFFFFSGR 0.150 132 FSQTVGEVF 0.150 488ISAFIRTLR 0.150 163 QQELCPSFL 0.135 199 TTIQQGISG 0.125 485 FPLISAFIR0.125 607 VGGVGVLSF 0.125 134 QTVGEVFYT 0.125 575 KNAFMLLMR 0.125 266LGVLAYGIY 0.125 40 LFLLFILGY 0.125 196 TNDTTIQQG 0.125 610 VGVLSFFFF0.125 360 VTFVLLLIC 0.125 156 MTVITSLQQ 0.125 677 NNGSLDRPY 0.125 498HTGSLAFGA 0.125 172 LPSAPALGR 0.125 195 ITNDTTIQQ 0.125 452 WTLNWVLAL0.125 353 STMFYPLVT 0.125 443 QIYGVLGLF 0.100 543 CCLWCLEKF 0.100 207GLIDSLNAR 0.100 407 SCNPTAHLV 0.100 180 RCFPWTNVT 0.100 354 TMFYPLVTF0.100

TABLE VIII V3-HLA-A1-9mers- 24P4C12 Each peptide is a portion of SEQ IDNO: 7; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 9 ITPPALPGI 0.500 8 NITPPALPG 0.5002 RCFPWTNIT 0.100 6 WTNITPPAL 0.050 7 TNITPPALP 0.001 1 GRCFPWTNI 0.0013 CFPWTNITP 0.000 5 PWTNITPPA 0.000 4 FPWTNITPP 0.000

TABLE VIII V5-HLA-A1-9mers- 24P4C12 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 1 VLEAILLLV 4.500 6 LLLVLIFLR 0.5004 AILLLVLIF 0.500 8 LVLIFLRQR 0.100 7 LLVLIFLRQ 0.050 5 ILLLVLIFL 0.0503 EAILLLVLI 0.020 9 VLIFLRQRI 0.010 2 LEAILLLVL 0.003

TABLE VIII V6-HLA-A1-9mers- 24P4C12 Each peptide is a portion of SEQ IDNO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 6 GLIPRSVFN 0.200 2 YSSKGLIPR 0.0755 KGLIPRSVF 0.025 7 LIPRSVFNL 0.005 3 SSKGLIPRS 0.003 4 SKGLIPRSV 0.0019 PRSVFNLQI 0.000 8 IPRSVFNLQ 0.000 1 GYSSKGLIP 0.000

TABLE VIII V7-HLA-A1-9mers- 24P4C12 Each peptide is a portion of SEQ IDNO: 15; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 7 VAVGQMMST 0.050 6 LVAVGQMMS 0.0508 AVGQMMSTM 0.010 5 ILVAVGQMM 0.010 4 WILVAVGQM 0.010 3 YWILVAVGQ 0.0011 SWYWILVAV 0.001 2 WYWILVAVG 0.000

TABLE VIII V8-HLA-A1-9mers- 24P4C12 Each peptide is a portion of SEQ IDNO: 17; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 11 PITPTGHVF 0.100 19 FQTSILGAY0.075 20 QTSILGAYV 0.050 17 HVFQTSILG 0.050 12 ITPTGHVFQ 0.050 1NYYWLPIMR 0.025 13 TPTGHVFQT 0.013 8 MRNPITPTG 0.010 4 WLPIMRNPI 0.010 5LPIMRNPIT 0.005 18 VFQTSILGA 0.003 10 NPITPTGHV 0.003 15 TGHVFQTSI 0.0039 RNPITPTGH 0.003 14 PTGHVFQTS 0.003 7 IMRNPITPT 0.001 3 YWLPIMRNP 0.00116 GHVFQTSIL 0.001 2 YYWLPIMRN 0.000 6 PIMRNPITP 0.000

TABLE VIII V9-HLA-A1-9mers- 24P4C12 Each peptide is a portion of SEQ IDNO: 19; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 11 PTQPATLGY 6.250 4 MTALYPLPT 0.12515 ATLGYVLWA 0.125 8 YPLPTQPAT 0.050 5 TALYPLPTQ 0.020 2 WAMTALYPL 0.02016 TLGYVLWAS 0.010 6 ALYPLPTQP 0.010 13 QPATLGYVL 0.005 17 LGYVLWASN0.005 10 LPTQPATLG 0.003 9 PLPTQPATL 0.002 14 PATLGYVLW 0.002 12TQPATLGYV 0.002 3 AMTALYPLP 0.001 18 GYVLWASNI 0.001 7 LYPLPTQPA 0.001 1YWAMTALYP 0.000

TABLE IX-V1 HLA-A1-10mers-24P4C12 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 594 VTDLLLFFGK 125.000 32 CTDVICCVLF25.000 120 CPEDPWTVGK 9.000 518 ILEYIDHKLR 9.000 680 SLDRPYYMSK 5.000698 KNEAPPDNKK 4.500 318 VLEAILLLML 4.500 488 ISAFIRTLRY 3.750 39VLFLLFILGY 2.500 262 ILGVLGVLAY 2.500 362 FVLLLICIAY 2.500 136VGEVFYTKNR 2.250 221 IFEDFAQSWY 2.250 700 EAPPDNKKRK 2.000 9 DDEAYGKPVK1.800 6 RDEDDEAYGK 1.800 417 SSCPGLMCVF 1.500 132 FSQTVGEVFY 1.500 134QTVGEVFYTK 1.000 469 FASFYWAFHK 1.000 369 IAYWAMTALY 1.000 378YLATSGQPQY 1.000 670 FLEDLERNNG 0.900 103 VAENGLQCPT 0.900 277WEEYRVLRDK 0.900 242 LSLLFILLLR 0.750 163 QQELCPSFLL 0.675 58 YGDPRQVLYP0.625 266 LGVLAYGIYY 0.625 348 VGQMMSTMFY 0.625 171 LLPSAPALGR 0.500 507LILTLVQIAR 0.500 237 GVALVLSLLF 0.500 320 EAILLLMLIF 0.500 208LIDSLNARDI 0.500 609 GVGVLSFFFF 0.500 353 STMFYPLVTF 0.500 464VLAGAFASFY 0.500 322 ILLLMLIFLR 0.500 35 VICCVLFLLF 0.500 606 VVGGVGVLSF0.500 521 YIDHKLRGVQ 0.500 662 CVDTLFLCFL 0.500 661 MCVDTLFLCF 0.500 265VLGVLAYGIY 0.500 49 IVVGIVAWLY 0.500 667 FLCFLEDLER 0.500 407 SCNPTAHLVN0.500 165 ELCPSFLLPS 0.500 77 MGENKDKPYL 0.450 547 CLEKFIKFLN 0.450 337AIALLKEASK 0.400 512 VQIARVILEY 0.375 689 KSLLKILGKK 0.300 305VQETWLAALI 0.270 18 KYDPSFRGPI 0.250 76 GMGENKDKPY 0.250 557 RNAYIMIAIY0.250 590 VLDKVTDLLL 0.250 677 NNGSLDRPYY 0.250 578 FMLLMRNIVR 0.250 187VTPPALPGIT 0.250 463 CVLAGAFASF 0.200 516 RVILEYIDHK 0.200 74 YCGMGENKDK0.200 72 GAYCGMGENK 0.200 423 MCVFQGYSSK 0.200 621 RIPGLGKDFK 0.200 170FLLPSAPALG 0.200 211 SLNARDISVK 0.200 161 SLQQELCPSF 0.200 253VAGPLVLVLI 0.200 186 NVTPPALPGI 0.200 618 FSGRIPGLGK 0.150 173PSAPALGRCF 0.150 118 SSCPEDPWTV 0.150 125 WTVGKNEFSQ 0.125 676RNNGSLDRPY 0.125 608 GGVGVLSFFF 0.125 286 KGASISQLGF 0.125 80 NKDKPYLLYF0.125 360 VTFVLLLICI 0.125 196 TNDTTIQQGI 0.125 198 DTTIQQGISG 0.125 293LGFTTNLSAY 0.125 271 YGIYYCWEEY 0.125 382 SGQPQYVLWA 0.125 467GAFASFYWAF 0.100 487 LISAFIRTLR 0.100 650 VIASGFFSVF 0.100 64 VLYPRNSTGA0.100 347 AVGQMMSTMF 0.100 272 GIYYCWEEYR 0.100 333 RIRIAIALLK 0.100 612VLSFFFFSGR 0.100 147 FCLPGVPWNM 0.100 216 DISVKIFEDF 0.100 53 IVAWLYGDPR0.100 326 MLIFLRQRIR 0.100 544 CLWCLEKFIK 0.100

TABLE IX-V3 HLA-A1-10mers-24P4C12 Each peptide is a portion of SEQ IDNO: 7; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 10 ITPPALPGIT 0.250 9 NITPPALPGI0.200 3 RCFPWTNITP 0.050 8 TNITPPALPG 0.013 7 WTNITPPALP 0.005 5FPWTNITPPA 0.001 2 GRCFPWTNIT 0.001 1 LGRCFPWTNI 0.000 6 PWTNITPPAL0.000 4 CFPWTNITPP 0.000

TABLE IX-V5 HLA-A1-10mers-24P4C12 Each peptide is a portion of SEQ IDNO: 11; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 2 VLEAILLLVL 4.500 6 ILLLVLIFLR 0.5004 EAILLLVLIF 0.500 8 LLVLIFLRQR 0.100 10 VLIFLRQRIR 0.100 7 LLLVLIFLRQ0.050 1 AVLEAILLLV 0.050 5 AILLLVLIFL 0.050 9 LVLIFLRQRI 0.010 3LEAILLLVLI 0.001

TABLE IX-V6 HLA-A1-10mers-24P4C12 Each peptide is a portion of SEQ IDNO: 13; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 7 GLIPRSVFNL 0.500 2 GYSSKGLIPR 0.0256 KGLIPRSVFN 0.005 5 SKGLIPRSVF 0.005 3 YSSKGLIPRS 0.003 10 PRSVFNLQIY0.003 4 SSKGLIPRSV 0.002 9 IPRSVFNLQI 0.001 1 QGYSSKGLIP 0.001 8LIPRSVFNLQ 0.001

TABLE IX-V7 HLA-A1-10mers-24P4C12 Each peptide is a portion of SEQ IDNO: 15; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 9 AVGQMMSTMF 0.100 6 ILVAVGQMMS 0.0507 LVAVGQMMST 0.050

TABLE IX-V7 HLA-A1-10mers-24P4C12 Each peptide is a portion of SEQ IDNO: 15; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 8 VAVGQMMSTM 0.010 5 WILVAVGQMM 0.0101 QSWYWILVAV 0.003 2 SWYWILVAVG 0.001 4 YWILVAVGQM 0.001 3 WYWILVAVGQ0.000

TABLE IX-V8 HLA-A1-10mers-24P4C12 Each peptide is a portion of SEQ IDNO: 17; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 1 LNYYWLPIMR 0.125 13 ITPTGHVFQT0.125 21 QTSILGAYVI 0.050 18 HVFQTSILGA 0.050 11 NPITPTGHVF 0.025 19VFQTSILGAY 0.025 12 PITPTGHVFQ 0.020 5 WLPIMRNPIT 0.020 4 YWLPIMRNPI0.005 9 MRNPITPTGH 0.005 20 FQTSILGAYV 0.003 15 PTGHVFQTSI 0.003 14TPTGHVFQTS 0.003 10 RNPITPTGHV 0.003 2 NYYWLPIMRN 0.003 16 TGHVFQTSIL0.003 17 GHVFQTSILG 0.003 6 LPIMRNPITP 0.001 8 IMRNPITPTG 0.001 7PIMRNPITPT 0.000 3 YYWLPIMRNP 0.000

TABLE IX-V9 HLA-A1-10mers-24P4C12 Each peptide is a portion of SEQ IDNO: 19; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 11 LPTQPATLGY 0.625 7 ALYPLPTQPA0.100 9 YPLPTQPATL 0.050 5 MTALYPLPTQ 0.050 12 PTQPATLGYV 0.025 4AMTALYPLPT 0.025 16 ATLGYVLWAS 0.025 17 TLGYVLWASN 0.020 15 PATLGYVLWA0.005 14 QPATLGYVLW 0.005 13 TQPATLGYVL 0.003 18 LGYVLWASNI 0.003 3WAMTALYPLP 0.002 2 YWAMTALYPL 0.001 10 PLPTQPATLG 0.001 6 TALYPLPTQP0.001 8 LYPLPTQPAT 0.001 19 GYVLWASNIS 0.001 1 AYWAMTALYP 0.000

TABLE X-V1 HLA-A0201-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 449 GLFWTLNWV 3255.381 322 ILLLMLIFL1699.774 580 LLMRNIVRV 1006.209 597 LLLFFGKLL 510.604 544 CLWCLEKFI476.257 598 LLFFGKLLV 437.482 170 FLLPSAPAL 363.588 86 LLYFNIFSC 360.526578 FMLLMRNIV 350.529 244 LLFILLLRL 309.050 41 FLLFILGYI 292.008 95ILSSNIISV 271.948 260 VLILGVLGV 271.948 56 WLYGDPRQV 204.761 42LLFILGYIV 179.368 650 VIASGFFSV 179.161 564 AIYGKNFCV 177.497 239ALVLSLLFI 131.975 604 LLVVGGVGV 118.238 589 VVLDKVTDL 110.872 268VLAYGIYYC 106.837 456 WVLALGQCV 103.580 537 IMCCFKCCL 99.667 446GVLGLFWTL 98.554 257 LVLVLILGV 88.043 660 GMCVDTLFL 84.856 686 YMSKSLLKI79.718 177 ALGRCFPWT 77.873 211 SLNARDISV 69.552 107 GLQCPTPQV 69.552241 VLSLLFILL 69.001 434 LIQRSVFNL 66.613 35 VICCVLFLL 66.613 547CLEKFIKFL 65.721 317 AVLEAILLL 65.219 240 LVLSLLFIL 64.306 302 YQSVQETWL54.798 309 WLAALIVLA 52.561 351 MMSTMFYPL 49.834 365 LLICIAYWA 46.451 45ILGYIVVGI 40.792 638 WLPIMTSIL 40.289 49 IVVGIVAWL 40.197 38 CVLFLLFIL37.827 148 CLPGVPWNM 37.260 232 ILVALGVAL 36.316 291 SQLGFTTNL 30.453 85YLLYFNIFS 26.508 506 ALILTLVQI 23.995 252 LVAGPLVLV 23.795 233 LVALGVALV23.795 525 KLRGVQNPV 18.501 339 ALLKEASKA 18.382 265 VLGVLAYGI 17.736326 MLIFLRQRI 17.736 340 LLKEASKAV 16.967 445 YGVLGLFWT 16.418 315VLAVLEAIL 14.890 457 VLALGQCVL 14.890 509 LTLVQIARV 13.975 119 SCPEDPWTV13.961 366 LICIAYWAM 13.064 226 AQSWYWILV 11.988 452 WTLNWVLAL 11.615426 FQGYSSKGL 9.963 554 FLNRNAYIM 9.370 642 MTSILGAYV 9.032 164QELGPSFLL 8.914 693 KILGKKNEA 8.846 251 RLVAGPLVL 8.759 501 SLAFGALIL8.759 487 LISAFIRTL 8.729 442 LQIYGVLGL 8.469 262 ILGVLGVLA 8.446 521YIDHKLRGV 8.094 373 AMTALYLAT 8.073 242 LSLLFILLL 7.666 134 QTVGEVFYT7.594 191 ALPGITNDT 7.452 590 VLDKVTDLL 7.118 362 FVLLLICIA 6.977 200KPYLLYFNI 6.756 83 KPYLLYFNI 6.636 314 IVLAVLEAI 6.471 383 GQPQYVLWA6.372 225 FAQSWYWIL 6.295 289 SISQLGFTT 5.943 364 LLLICIAYW 5.929 596DLLLFFGKL 5.564 611 GVLSFFFFS 5.557 282 VLRDKGASI 5.526 154 WNMTVITSL5.459 380 ATSGQPQYV 5.313 612 VLSFFFFSG 5.305 100 IISVAENGL 4.993 158VITSLQQEL 4.993 504 FGALILTLV 4.804 536 CIMCCFKCC 4.802 246 FILLLRLVA4.767 357 YPLVTFVLL 4.510

TABLE X-V3 HLA-A0201-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 7; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 6 WTNITPPAL 1.365 9 ITPPALPGI 0.5672 RCFPWTNIT 0.074 8 NITPPALPG 0.010 4 FPWTNITPP 0.009 1 GRCFPWTNI 0.0027 TNITPPALP 0.000 5 PWTNITPPA 0.000 3 CFPWTNITP 0.000

TABLE X-V5 HLA-A0201-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 11; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 5 ILLLVLIFL 1699.774 9 VLIFLRQRI17.736 1 VLEAILLLV 5 6 LLLVLIFLR 1.251 2 LEAILLLVL 0.666 7 LLVLIFLRQ0.048 4 AILLLVLIF 0.036 3 EAILLLVLI 0.025 8 LVLIFLRQR 0.014

TABLE X-V6 HLA-A0201-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 7 LIPRSVFNL 66.613 6 GLIPRSVFN 0.4104 SKGLIPRSV 0.019 5 KGLIPRSVF 0.003 2 YSSKGLIPR 0.001 9 PRSVFNLQI 0.0003 SSKGLIPRS 0.000 8 IPRSVFNLQ 0.000 1 GYSSKGLIP 0.000

TABLE X-V7 HLA-A0201-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 15; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 5 ILVAVGQMM 8.446 4 WILVAVGQM 3.4768 AVGQMMSTM 1.000 7 VAVGQMMST 0.405 1 SWYWILVAV 0.071 6 LVAVGQMMS 0.0112 WYWILVAVG 0.000 3 YWILVAVGQ 0.000

TABLE X-V8 HLA-A0201-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 17; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 4 WLPIMRNPI 47.991 20 QTSILGAYV5.313 7 IMRNPITPT 1.599 13 TPTGHVFQT 0.649 15 TGHVFQTSI 0.259 10NPITPTGHV 0.059 5 LPIMRNPIT 0.034 18 VFQTSILGA 0.013 19 FQTSILGAY 0.01016 GHVFQTSIL 0.006 12 ITPIGHVFQ 0.002 2 YYWLPlMRN 0.001 17 HVFQTSILG0.001 9 RNPITPTGH 0.000 6 PIMRNPITP 0.000 11 PITPTGHVF 0.000 14PTGHVFQTS 0.000 8 MRNPITPTG 0.000 3 YWLPIMRNP 0.000 1 NYYWLPIMR 0.000

TABLE X-V9 HLA-A0201-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 19; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 2 WAMTALYPL 11.615 12 TQPATLGYV11.597 15 ATLGYVLWA 3.230 16 TLGYVLWAS 1.285 8 YPLPTQPAT 0.828 9PLPTQPATL 0.470 4 MTALYPLPT 0.176 13 QPATLGYVL 0.057 6 ALYPLPTQP 0.048 3AMTALYPLP 0.016 17 LGYVLWASN 0.004 5 TALYPLPTQ 0.002 18 GYVLWASNI 0.0017 LYPLPTQPA 0.001 10 LPTQPATLG 0.001 1 YWAMTALYP 0.000 14 PATLGYVLW0.000 11 PTQPATLGY 0.000

TABLE XI-V1-HLA-A0201-10 mers-24P4C12 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 354 TMFYPLVTFV 2351.109  85YLLYFNIFSC 1127.969 579 MLLMRNIVRV 1006.209 603 KLLVVGGVGV 900.698 309WLAALIVLAV 735.860 351 MMSTMFYPLV 486.748  41 FLLFILGYIV 485.348 641IMTSILGAYV 469.669 546 WCLEKFIKFL 467.771 597 LLLFFGKLLV 437.482 598LLFFGKLLVV 412.546 665 TLFLCFLEDL 338.500 241 VLSLLFILLL 317.403 649YVIASGFFSV 308.501 433 GLIQRSVFNL 284.974 508 ILTLVQIARV 271.948 232ILVALGVALV 271.948  42 LLFILGYIVV 269.051 339 ALLKEASKAV 257.342 449GLFWTLNWVL 243.051 244 LLFILLLRLV 201.242 243 SLLFILLLRL 181.794 364LLLICIAYWA 171.868  48 YIVVGIVAWL 170.923 251 RLVAGPLVLV 159.970 321AILLLMLIFL 137.482  56 WLYGDPRQVL 128.926 239 ALVSLLFIL 116.840 350QMMSTMFYPL 108.462  86 LLYFNIFSCI 107.833 365 LLICIAYWAM 95.013 259LVLILGVLGV 88.043 162 LQQELCPSFL 83.030 580 LLMRNIVRVV 82.509  94CILSSNIISV 81.385 517 VILEYIDHKL 75.751 554 FLNRNAYIMI 71.986 686YMSKSLLKIL 66.925  44 FILGYIVVGI 56.155 133 SQTVGEVFYT 55.435 438SVFNLQIYGV 51.790 231 WILVALGVAL 49.993 235 ALGVALVLSL 49.134 441NLQIYGVLGL 49.134 660 GMCVDTLFLC 47.864 325 LMLIFLRQRI 47.394 536CIMCCFKCCL 41.299 315 VLAVLEAILL 36.316 448 LGLFWTLNWV 36.126 662CVDTLFLCFL 35.941  64 VLYPRNSTGA 27.026 589 VVLDKVTDLL 23.620 596DLLLFFGKLL 22.527 240 LVLSLLFILL 22.339 357 YPLVTFVLLL 20.744 267GVLAYGIYYC 20.346 304 SVQETWLAAL 17.627 248 LLLRLVAGPL 17.468 302YQSVQETWLA 17.378 501 SLAFGALILT 17.140 317 AVLEAILLLM 15.167 590VLDKVTDLLL 14.526  45 ILGYIVVGIV 14.495 659 FGMCVDTLFL 13.054 456WVLALGQCVL 13.044 148 CLPGVPWNMT 12.668 108 LQCPTPQVCV 11.988 478KPQDIPTFPL 11.606 238 VALVLSLLFI 11.529 312 ALIVLAVLEA 11.426 459ALGQCVLAGA 11.426 571 CVSAKNAFML 10.841 563 IAIYGKNFCV 9.525 445YGVLGLFWTL 9.141 379 LATSGQPQYV 9.032 327 LIFLRQRIRI 9.023 249LLRLVAGPLV 8.986 539 CCFKCCLWCL 8.900 513 QIARVILEYI 8.892 510TLVQIARVIL 8.759 457 VLALGQCVLA 8.446  95 ILSSNIISVA 7.964 657SVFGMCVDTL 7.794 225 FAQSWYWILV 7.554 588 VVVLDKVTDL 7.309 593KVTDLLLFFG 6.865 368 CIAYWAMTAL 6.756 562 MIAIYGKNFC 6.387 363VLLLICIAYW 5.929  36 ICCVLFLLFI 5.565 318 VLEAILLLML 5.346 292QLGFTTNLSA 4.968 314 IVLAVLEAIL 4.821 393 NISSPGCEKV 4.686 506ALILTLVQIA 4.685 260 VLILGVLGVL 4.452 604 LLVVGGVGVL 4.452 261LILGVLGVLA 4.297 502 LAFGALILTL 4.292 147 FCLPGVPWNM 4.140

TABLE XI-V3-HLA-A0201-10 mers-24P4C12 Each peptide is a portion of SEQID NO: 7; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score  9 NITPPALPGI 3.299  5 FPWTNITPPA1.238  1 LGRCFPWTNI 0.015 10 ITPPALPGIT 0.009  7 WTNITPPALP 0.001  8TNITPPALPG 0.000  2 GRCFPWTNIT 0.000  3 RCFPWTNITP 0.000  6 PWTNITPPAL0.000  4 CFPWTNITPP 0.000

TABLE XI-V5-HLA-A0201-10 mers-24P4C12 Each peptide is a portion of SEQID NO: 11; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score  1 AVLEAILLLV 212.340  5 AILLLVLIFL137.482  9 LVLIFLRQRI 5.742  2 VLEAILLLVL 2.192  6 ILLLVLIFLR 1.251  3LEAILLLVLI 0.793  7 LLLVLIFLRQ 0.178  8 LLVLIFLRQR 0.044 10 VLIFLRQRIR0.002  4 EAILLLVLIF 0.000

TABLE XI-V6-HLA-A0201-10 mers-24P4C12 Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score  7 GLIPRSVFNL 284.974  6 KGLIPRSVFN0.035  9 IPRSVFNLQI 0.033  8 LIPRSVFNLQ 0.007  3 YSSKGLIPRS 0.005  4SSKGLIPRSV 0.003  1 QGYSSKGLIP 0.000  5 SKGLIPRSVF 0.000  2 GYSSKGLIPR0.000 10 PRSVFNLQIY 0.000

TABLE XI-V7-HLA-A0201-10 mers-24P4C12 Each peptide is a portion of SEQID NO: 15; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 5 WILVAVGQMM 11.626 1 QSWYWILVAV8.667 7 LVAVGQMMST 2.550 8 VAVGQMMSTM 0.270 6 ILVAVGQMMS 0.127 9AVGQMMSTMF 0.007 4 YWILVAVGQM 0.001 3 WYWILVAVGQ 0.000 2 SWYWILVAVG0.000

TABLE XI-V8-HLA-A0201-10 mers-24P4C12 Each peptide is a portion of SEQID NO: 17; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 20 FQTSILGAYV 178.411  5 WLPIMRNPIT14.054 13 ITPTGHVFQT 2.347  7 PIMRNPITPT 0.192 18 HVFQTSILGA 0.126 21QTSILGAYVI 0.059 10 RNPITPTGHV 0.059 16 TGHVFQTSIL 0.057  4 YWLPIMRNPI0.025 15 PTGHVFQTSI 0.012  8 IMRNPITPTG 0.007 14 TPTGHVFQTS 0.001  1LNYYWLPIMR 0.001 12 PITPTGHVFQ 0.000 11 NPITPTGHVF 0.000  6 LPIMRNPITP0.000  2 NYYWLPIMRN 0.000 17 GHVFQTSILG 0.000  3 YYWLPIMRNP 0.000 19VFQTSILGAY 0.000  9 MRNPITPTGH 0.000

TABLE XI-V9-HLA-A0201-10 mers-24P4C12 Each peptide is a portion of SEQID NO: 19; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score  7 ALYPLPTQPA 15.898  4 AMTALYPLPT5.382  9 YPLPTQPATL 2.373 13 TQPATLGYVL 0.888 18 LGYVLWASNI 0.370 17TLGYVLWASN 0.127 16 ATLGYVLWAS 0.066 12 PTQPATLGYV 0.035  2 YWAMTALYPL0.031 15 PATLGYVLWA 0.019  3 WAMTALYPLP 0.005  8 LYPLPTQPAT 0.002 10PLPTQPATLG 0.002 11 LPTQPATLGY 0.001  5 MTALYPLPTQ 0.001  6 TALYPLPTQP0.001 14 QPATLGYVLW 0.001  1 AYWAMTALYP 0.000 19 GYVLWASNIS 0.000

TABLE XII-V1-HLA-A3-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 421 GLMCVFQGY 81.000 135 TVGEVFYTK40.500 207 GLIDSLNAR 27.000 323 LLLMLIFLR 27.000 243 SLLFILLLR 27.000354 TMFYPLVTF 22.500 690 SLLKILGKK 20.250 517 VILEYIDHK 20.250 363VLLLICIAY 18.000 585 IVRVVVLDK 18.000 560 YIMIAIYGK 13.500 508 ILTLVQIAR12.000 579 MLLMRNIVR 12.000 267 GVLAYGIYY 10.800 424 CVFQGYSSK 10.000244 LLFILLLRL 9.000 464 VLAGAFASF 9.000 272 GIYYCWEEY 6.000 351MMSTMFYPL 5.400 470 ASFYWAFHK 4.500 449 GLFWTLNWV 4.500  86 LLYFNIFSC4.500 446 GVLGLFWTL 3.645 660 GMCVDTLFL 3.600 633 HLNYYWLPI 3.600 542KCCLWCLEK 3.600 241 VLSLLFILL 3.600  42 LLFILGYIV 3.000 393 NISSPGCEK3.000 325 LMLIFLRQR 2.700  45 ILGYIVVGI 2.700 322 ILLLMLIFL 2.700 239ALVLSLLFI 2.700 641 IMTSILGAY 2.700 598 LLFFGKLLV 2.000 260 VLILGVLGV1.800 265 VLGVLAYGI 1.800 513 QIARVILEY 1.800 609 GVGVLSFFF 1.800 537IMCCFKCCL 1.800  50 VVGIVAWLY 1.800 686 YMSKSLLKI 1.800 251 RLVAGPLVL1.800 593 KVTDLLLFF 1.800 358 PLVTFVLLL 1.620 544 CLWCLEKFI 1.500 689KSLLKILGK 1.350 525 KLRGVQNPV 1.350 170 FLLPSAPAL 1.350 547 CLEKFIKFL1.350 597 LLLFFGKLL 1.350 365 LLICIAYWA 1.350 506 ALILTLVQI 1.350 148CLPGVPWNM 1.350 501 SLAFGALIL 1.200 662 CVDTLFLCF 1.200 349 GQMMSTMFY1.080 443 QIYGVLGLF 1.012 321 AILLLMLIF 0.900 590 VLDKVTDLL 0.900 326MLIFLRQRI 0.900 268 VLAYGIYYC 0.900 107 GLQCPTPQV 0.900 613 LSFFFFSGR0.900 318 VLEAILLLM 0.900 232 ILVALGVAL 0.900 518 ILEYIDHKL 0.900 452WTLNWVLAL 0.810 596 DLLLFFGKL 0.729 645 ILGAYVIAS 0.720 258 VLVLILGVL0.608  49 IVVGIVAWL 0.608  41 FLLFILGYI 0.608  54 VAWLYGDPR 0.600 665TLFLCFLED 0.600  95 ILSSNIISV 0.600 457 VLALGQCVL 0.600 282 VLRDKGASI0.600 554 FLNRNAYIM 0.600  39 VLFLLFILG 0.600 315 VLAVLEAIL 0.600 638WLPIMTSIL 0.600 434 LIQRSVFNL 0.540 612 VLSFFFFSG 0.540 611 GVLSFFFFS0.486 647 GAYVIASGF 0.450 580 LLMRNIVRV 0.450 364 LLLICIAYW 0.450 564AIYGKNFCV 0.450 237 GVALVLSLL 0.405  38 CVLFLLFIL 0.405 204 GISGLIDSL0.405  35 VICCVLFLL 0.405 317 AVLEAILLL 0.405 240 LVLSLLFIL 0.405 668LCFLEDLER 0.400 388 VLWASNISS 0.400 489 SAFIRTLRY 0.400 211 SLNARDISV0.400  85 YLLFNIFS 0.360

TABLE XII V3-HLA-A3-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 7; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 9 ITPPALPGI 0.068 6 WTNITPPAL 0.0302 RCFPWTNIT 0.022 8 NITPPALPG 0.009 1 GRCFPWTNI 0.003 4 FPWTNITPP 0.0027 TNITPPALP 0.000 3 CFPWTNITP 0.000 5 PWTNITPPA 0.000

TABLE XII-V5-HLA-A3-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 11; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 6 LLLVLIFLR 27.000 5 ILLLVLIFL 4.0504 AILLLVLIF 1.800 9 VLIFLRQRI 0.900 1 VLEAILLLV 0.900 7 LLVLIFLRQ 0.2708 LVLIFLRQR 0.270 2 LEAILLLVL 0.005 3 EAILLLVLI 0.004

TABLE XII-V6-HLA-A3-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 7 LIPRSVFNL 0.540 6 GLIPRSVFN 0.1352 YSSKGLIPR 0.060 5 KGLIPRSVF 0.013 8 IPRSVFNLQ 0.001 3 SSKGLIPRS 0.0009 PRSVFNLQI 0.000 1 GYSSKGLIP 0.000 4 SKGLIPRSV 0.000

TABLE XII-V7-HLA-A3-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 15; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 5 ILVAVGQMM 0.450 8 AVGQMMSTM 0.0304 WILVAVGQM 0.027 6 LVAVGQMMS 0.008 7 VAVGQMMST 0.007 1 SWYWILVAV 0.0022 WYWILVAVG 0.000 3 YWILVAVGQ 0.000

TABLE XII-V8-HLA-A3-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 17; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score  4 WLPIMRNPI 0.600  7 IMRNPITPT0.225 19 FQTSILGAY 0.081  1 NYYWLPIMR 0.040 11 PITPTGHVF 0.030 17HVFQTSILG 0.020 13 TPTGHVFQT 0.013 20 QTSILGAYV 0.010 16 GHVFQTSIL 0.00315 TGHVFQTSI 0.002  5 LPIMRNPIT 0.002 10 NPITPTGHV 0.001 12 ITPTGHVFQ0.001 14 PTGHVFQTS 0.001 18 VFQTSILGA 0.001  6 PIMRNPITP 0.001  2YYWLPIMRN 0.000  9 RNPITPTGH 0.000 8 MRNPITPTG 0.000 3 YWLPIMRNP 0.000

TABLE XII-V9-HLA-A3-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 19; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 15 ATLGYVLWA 0.405 16 TLGYVLWAS0.270  6 ALYPLPTQP 0.150 11 PTQPATLGY 0.060  9 PLPTQPATL 0.060  2WAMTALYPL 0.041  4 MTALYPLPT 0.030  3 AMTALYPLP 0.020 13 QPATLGYVL 0.01818 GYVLWASNI 0.008 12 TQPATLGYV 0.003  8 YPLPTQPAT 0.002  5 TALYPLPTQ0.001  7 LYPLPTQPA 0.000 10 LPTQPATLG 0.000 14 PATLGYVLW 0.000 17LGYVLWASN 0.000  1 YWAMTALLYP 0.000

TABLE XIII-V1-HLA-A3-10 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 544 CLWCLEKFIK 300.000  39 VLFLLFILGY180.000 680 SLDRPYYMSK 120.000 612 VLSFFFFSGR 36.000 134 QTVGEVFYTK30.375 211 SLNARDISVK 30.000 449 GLFWTLNWVL 27.000 322 ILLLMLIFLR 27.000584 NIVRVVVLDK 27.000 433 GLIQRSVFNL 24.300 262 ILGVLGVLAY 24.000 272GIYYCWEEYR 18.000 464 VLAGAFASFY 18.000 665 TLFLCFLEDL 13.500 516RVILEYIDHK 13.500  86 LLYFNIFSCI 13.500 171 LLPSAPALGR 12.000 578FMLLMRNIVR 12.000  76 GMGENKDKPY 9.000 594 VTDLLLFFGK 9.000 350QMMSTMFYPL 8.100 667 FLCFLEDLER 8.000  56 WLYGDPRQVL 6.750 333RIRIAIALLK 6.000 609 GVGVLSFFFF 5.400 241 VLSLLFILLL 5.400 561IMIAIYGKNF 4.500 239 ALVLSLLFIL 4.050  49 IVVGIVAWLY 4.050 378YLATSGQPQY 4.000 441 NLQIYGVLGL 3.600 235 ALGVALVLSL 3.600 598LLFFGKLLVV 3.000 621 RIPGLGKDFK 3.000 354 TMFYPLVTFV 3.000  72GAYCGMGENK 3.000 324 LLMLIFLRQR 2.700 660 GMCVDTLFLC 2.700 467GAFASFYWAF 2.700 243 SLLFILLLRL 2.700  83 KPYLLYFNIF 2.700  42LLFILGYIVV 2.000 518 ILEYIDHKLR 2.000 161 SLQQELCPSF 2.000 337AIALLKEASK 2.000 362 FVLLLICIAY 1.800 650 VIASGFFSVF 1.800 606VVGGVGVLSF 1.800 507 LILTLVQIAR 1.800 329 FLRQRIRIAI 1.800 318VLEAILLLML 1.800 624 GLGKDFKSPH 1.800 309 WLAALIVLAV 1.800 312ALIVLAVLEA 1.800 469 FASFYWAFHK 1.800  64 VLYPRNSTGA 1.500 364LLLICIAYWA 1.350 657 SVFGMCVDTL 1.350  85 YLLYFNIFSC 1.350 220KIFEDFAQSW 1.350 264 GVLGVLAYGI 1.215 315 VLAVLEAILL 1.200 237GVALVLSLLF 1.200 554 FLNRNAYIMI 1.200 590 VLDKVTDLLL 1.200 265VLGVLAYGIY 1.200  35 VICCVLFLLF 1.200  53 IVAWLYGDPR 1.200 447VLGLFWTLNW 1.200 268 VLAYGIYYCW 0.900 413 HLVNSSCPGL 0.900 275YCWEEYRVLR 0.900 232 ILVALGVALV 0.900 325 LMLIFLRQRI 0.900 463CVLAGAFASF 0.900 525 KLRGVQNPVA 0.900 506 ALILTLVQIA 0.900 603KLLVVGGVGV 0.900 633 HLNYYWLPIM 0.900 510 TLVQIARVIL 0.900 365LLICIAYWAM 0.900  41 FLLFILGYIV 0.900 512 VQIARVILEY 0.810 604LLVVGGVGVL 0.810 251 RLVAGPLVLV 0.675 260 VLILGVLGVL 0.608  44FILGYIVVGI 0.608 107 GLQCPTPQVC 0.600 327 LIFLRQRIRI 0.600 326MLIFLRQRIR 0.600 597 LLLFFGKLLV 0.600 487 LISAFIRTLR 0.600 120CPEDPWTVGK 0.600 351 MMSTMFYPLV 0.600 240 LVLSLLFILL 0.540 252LVAGPLVLVL 0.540 360 VTFVLLLICI 0.450 363 VLLLICIAYW 0.450 579MLLMRNIVRV 0.450  95 ILSSNIISVA 0.450

TABLE XIII-V3-HLA-A3-10 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 7; each start position is specified, the length of peptide is 10amino acids, and the end postion for each peptide is the start positionplus nine. Start Subsequence Score  9 NITPPALPGI 0.135  5 FPWTNITPPA0.015  3 RCFPWTNITP 0.003 10 ITPPALPGIT 0.002  7 WTNITPPALP 0.002  1LGRCFPQTNI 0.001  2 GRCFPWTNIT 0.001  8 TNITPPALPG 0.000  6 PWTNITPPAL0.000  4 CFPWTNITPP 0.000

TABLE XIII-V5-HLA-A3-10 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 11; each start position is specified, the length of peptide is 10amino acids, and the end postion for each peptide is the start positionplus nine. Start Subsequence Score  6 ILLLVLIFLR 27.000  8 LLVLIFLRQR2.700  2 VLEAILLLVL 1.800 10 VLIFLRQRIR 0.600  5 AILLLVLIFL 0.405  7LLLVLIFLRQ 0.270  1 AVLEAILLLV 0.203  9 LVLIFLRQRI 0.090  4 EAILLLVLIF0.054  3 LEAILLLVLI 0.003

TABLE XIlI-V6-HLA-A3-10 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 13; each start position is specified, the length of peptide is 10amino acids, and the end postion for each peptide is the start positionplus nine. Start Subsequence Score  7 GLIPRSVFNL 36.450  2 GYSSKGLIPR0.036  9 IPRSVFNLQI 0.036  8 LIPRSVFNLQ 0.009  5 SKGLIPRSVF 0.003 10PRSVFNLQIY 0.001  3 YSSKGLIPRS 0.000  4 SSKGLIPRSV 0.000  1 QGYSSKGLIP0.000  6 GKLIPRSVFN 0.000

TABLE XIII-V7-HLA-A3-10 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 15; each start position is specified, the length of peptide is 10amino acids, and the end postion for each peptide is the start positionplus nine. Start Subsequence Score 9 AVGQMMSTMF 0.200 6 ILVAVGQMMS 0.1205 WILVAVGQMM 0.045 7 LVAVGQMMST 0.030 1 QSWYWILVAV 0.011 8 VAVGQMMSTM0.007 2 SWYWILVAVG 0.000 4 YWILVAVGQM 0.000 3 WYWILVAVGQ 0.000

TABLE XIII-V8-HLA-A3-10 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 17; each start position is specified, the length of peptide is 10amino acids, and the end postion for each peptide is the start positionplus nine. Start Subsequence Score 18 HVFQTSILGA 0.300  5 WLPIMRNPIT0.100 21 QTSILGAYVI 0.090  1 LNYYWLPIMR 0.080 13 ITPTGHVFQT 0.045 11NPITPTGHVF 0.030  8 IMRNPITPTG 0.030 15 PTGHVFQTSI 0.009 20 FQTSILGAYV0.006  7 PIMRNPITPT 0.003 14 TPTGHVFQTS 0.003 19 VFQTSILGAY 0.003  4YWLPIMRNPI 0.001  6 LPIMRNPITP 0.001 16 TGHVFQTSIL 0.001  2 NYYWLPIMRN0.000  9 MRNPITPTGH 0.000 12 PITPTGHVFQ 0.000 17 GHVFQTSILG 0.000 10RNPITPTGHV 0.000  3 YYWLPIMRNP 0.000

TABLE XIII-V9-HLA-A3-10 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 19; each start position is specified, the length of peptide is 10amino acids, and the end postion for each peptide is the start positionplus nine. Start Subsequence Score  7 ALYPLPTQPA 2.250  4 AMTALYPLPT0.600 11 LPTQPATLGY 0.080 13 TQPATLGYVL 0.054 16 ATLGYVLWAS 0.030 17TLGYVLWASN 0.020  9 YPLPTQPATL 0.013 18 LGYVLWASNI 0.009 15 PATLGYVLWA0.004 10 PLPTQPATLG 0.003  2 YWAMTALYPL 0.003  5 MTALYPLPTQ 0.002 14QPATLGYVLW 0.002 12 PTQPATLGYV 0.001  6 TALYPLPTQP 0.000  3 WAMTALYPLP0.000  1 AYWAMTALYP 0.000 19 GYVLWASNIS 0.000  8 LYPLPTQPAT 0.000

TABLE XIV-V1-HLA-A1101-9 mers-24P4C12 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 9amino acids, and the end postion for each peptide is the start positionplus nine. Start Subsequence Score 135 TVGEVFYTK 4.000 585 IVRVVVLDK4.000 424 CVFQGYSSK 4.000 560 YIMIAIYGK 1.600 685 YYMSKSLLK 1.600 542KCCLWCLEK 1.200 690 SLLKILGKK 0.600 517 VILEYIDHK 0.600  73 AYCGMGENK0.400 393 NISSPGCEK 0.400 207 GLIDSLNAR 0.360 323 LLLMLIFLR 0.360 338IALLKEASK 0.300 579 MLLMRNIVR 0.240 243 SLLFILLLR 0.240 622 IPGLGKDFK0.200 689 KSLLKILGK 0.180 516 RVILEYIDH 0.180 609 GVGVLSFFF 0.180 485FPLISAFIR 0.180 446 GVLGLFWTL 0.180 267 GVLAYGIYY 0.180 273 IYYCWEEYR0.160 508 ILTLVQIAR 0.160 668 LCFLEDLER 0.160 698 KNEAPPDNK 0.120 470ASFYWAFHK 0.120 593 KVTDLLLFF 0.120 701 APPDNKKRK 0.100 595 TDLLLFFGK0.090  38 CVLFLLFIL 0.090 240 LVLSLLFIL 0.090  54 VAWLYGDPR 0.080 172LPSAPALGR 0.080 349 GQMMSTMFY 0.072 334 IRIAIALLK 0.060 545 LWCLEKFIK0.060 567 GKNFCVSAK 0.060 317 AVLEAILLL 0.060 699 NEAPPDNKK 0.060 151GVPWNMTVI 0.060 237 GVALVLSLL 0.060 257 LVLVLILGV 0.060  20 DPSFRGPIK0.060 575 KNAFMLLMR 0.048 212 LNARDISVK 0.040 359 LVTFVLLLI 0.040  16PVKYDPSFR 0.040 304 SVQETWLAA 0.040 619 SGRIPGLGK 0.040  50 VVGIVAWLY0.040 681 LDRPYYMSK 0.040 662 CVDTLFLCF 0.040   7 DEDDEAYGK 0.036  83KPYLLYFNI 0.036  47 GYIVVGIVA 0.036 251 RLVAGPLVL 0.036 383 GQPQYVLWA0.036  49 IVVGIVAWL 0.030 314 IVLAVLEAI 0.030 456 WVLALGQCV 0.030 589VVLDKVTDL 0.030 452 WTLNWVLAL 0.030 141 YTKNRNFCL 0.030 498 HTGSLAFGA0.030 605 LVVGGVGVL 0.030 362 FVLLLICIA 0.030 611 GVLSFFFFS 0.027 137GEVFYTKNR 0.027 564 AIYGKNFCV 0.024 272 GIYYCWEEY 0.024  60 DPRQVLYPR0.024 421 GLMCVFQGY 0.024 467 GAFASFYWA 0.024 449 GLFWTLNWV 0.024 660GMCVDTLFL 0.024 496 RYHTGSLAF 0.024 511 LVQIARVIL 0.020 218 SVKIFEDFA0.020 233 LVALGVALV 0.020  22 SFRGPIKNR 0.020  75 CGMGENKDK 0.020 414LVNSSCPGL 0.020 252 LVAGPLVLV 0.020 571 CVSAKNAFM 0.020 347 AVGQMMSTM0.020 534 ARCIMCCFK 0.020 527 RGVQNPVAR 0.018  34 DVICCVLFL 0.018 693KILGKKNEA 0.018 461 GQCVLAGAF 0.018   4 KQRDEDDEA 0.018 331 RQRIRIAIA0.018  10 DEAYGKPVK 0.018 442 LQIYGVLGL 0.018 255 GPLVLVLIL 0.018 598LLFFGKLLV 0.016  42 LLFILGYIV 0.016 244 LLFILLLRL 0.016 327 LIFLRQRIR0.016

TABLE XIV-V3-HLA-A1101-9 mers-24P4C12 Each peptide is a portion of SEQID NO: 7; each start position is specified, the length of peptide is 9amino acids, and the end postion for each peptide is the start positionplus eight. Start Subsequence Score 9 ITPPALPGI 0.010 6 WTNITPPAL 0.0102 RCFPWTNIT 0.001 8 NITPPALPG 0.001 1 GRCFPWTNI 0.001 4 FPWTNITPP 0.0003 CFPWTNITP 0.000 7 TNITPPALP 0.000 5 PWTNITPPA 0.000

TABLE XIV-V5-HLA-A1101-9 mers-24P4C12 Each peptide is a portion of SEQID NO: 11; each start position is specified, the length of peptide is 9amino acids, and the end postion for each peptide is the start positionplus eight. Start Subsequence Score 6 LLLVLIFLR 0.360 8 LVLIFLRQR 0.0604 AILLLVLIF 0.012 5 ILLLVLIFL 0.012 1 VLEAILLLV 0.008 9 VLIFLRQRI 0.0067 LLVLIFLRQ 0.001 2 LEAILLLVL 0.001 3 EAILLLVLI 0.001

TABLE XIV-V6-HLA-A1101-9 mers-24P4C12 Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 9amino acids, and the end postion for each peptide is the start positionplus eight. Start Subsequence Score 7 LIPRSVFNL 0.012 2 YSSKGLIPR 0.0081 GYSSKGLIP 0.002 6 GLIPRSVFN 0.002 5 KGLIPRSVF 0.001 8 IPRSVFNLQ 0.0009 PRSVFNLQI 0.000 4 SKGLIPRSV 0.000 3 SSKGLIPRS 0.000

TABLE XIV-V7-HLA-A1101-9 mers-24P4C12 Each peptide is a portion of SEQID NO: 15; each start position is specified, the length of peptide is 9amino acids, and the end postion for each peptide is the start positionplus eight. Start Subsequence Score 8 AVGQMMSTM 0.020 5 ILVAVGQMM 0.0064 WILVAVGQM 0.006 6 LVAVGQMMS 0.004 2 WYWILVAVG 0.001 7 VAVGQMMST 0.0011 SWYWILVAV 0.000 3 YWILVAVGQ 0.000

TABLE XIV-V8-HLA-A1101-9 mers-24P4C12 Each peptide is a portion of SEQID NO: 17; each start position is specified, the length of peptide is 9amino acids, and the end postion for each peptide is the start positionplus eight. Start Subsequence Score  1 NYYWLPIMR 0.320 20 QTSILGAYV0.010 17 HVFQTSILG 0.008 19 FQTSILGAY 0.006 18 VFQTSILGA 0.004  4WLPIMRNPI 0.004 10 NPITPTGHV 0.003  2 YYWLPIMRN 0.002  9 RNPITPTGH 0.00112 ITPTGHVFQ 0.001 16 GHVFQTSIL 0.001 13 TPTGHVFQT 0.001 11 PITPTGHVF0.000  7 IMRNPITPT 0.000  5 LPIMRNPIT 0.000 15 TGHVFQTSI 0.000  6PIMRNPITP 0.000 14 PTGHVFQTS 0.000  8 MRNPITPTG 0.000  3 YWLPIMRNP 0.000

TABLE XIV-V8-HLA-A1101-9 mers-24P4C12 Each peptide is a portion of SEQID NO: 19; each start position is specified, the length of peptide is 9amino acids, and the end postion for each peptide is the start positionplus eight. Start Subsequence Score 15 ATLGYVLWA 0.030 18 GYVLWASNI0.018  2 WAMTALYPL 0.008 12 TQPATLGYV 0.006  7 LYPLPTQPA 0.004 13QPATLGYVL 0.004  4 MTALYPLPT 0.002 11 PTQPATLGY 0.002  6 ALYPLPTQP 0.00116 TLGYVLWAS 0.001  3 AMTALYPLP 0.000  9 PLPTQPATL 0.000  8 YPLPTQPAT0.000  5 TALYPLPTQ 0.000 10 LPTQPATLG 0.000 14 PATLGYVLW 0.000 17LGYVLWASN 0.000  1 YWAMTALYP 0.000

TABLE XV-V1-A1101-10 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 10amino acids, and the end postion for each peptide is the start positionplus nine. Start Subsequence Score 516 RVILEYIDHK 9.000 594 VTDLLLFFGK3.000 134 QTVGEVFYTK 3.000 333 RIRIAIALLK 2.400 544 CLWCLEKFIK 2.400 621RIPGLGKDFK 1.200 559 AYIMIAIYGK 1.200  72 GAYCGMGENK 1.200 584NIVRVVVLDK 1.200 680 SLDRPYYMSK 0.800 469 FASFYWAFHK 0.600 272GIYYCWEEYR 0.480 428 GYSSKGLIQR 0.480 337 AIALLKEASK 0.400  53IVAWLYGDPR 0.400 211 SLNARDISVK 0.400 322 ILLLMLIFLR 0.360 423MCVFQGYSSK 0.300 507 LILTLVQIAR 0.240 578 FMLLMRNIVR 0.240 120CPEDPWTVGK 0.200 533 VARCIMCCFK 0.200  15 KPVKYDPSFR 0.180 264GVLGVLAYGI 0.180 609 GVGVLSFFFF 0.180 684 PYYMSKSLLK 0.160 667FLCFLEDLER 0.160 171 LLPSAPALGR 0.160   6 RDEDDEAYGK 0.120 698KNEAPPDNKK 0.120 484 TFPLISAFIR 0.120 237 GVALVLSLLF 0.120  74YCGMGENKDK 0.100 689 KSLLKILGKK 0.090 649 YVIASGFFSV 0.090 281RVLRDKGASI 0.090 612 VLSFFFFSGR 0.080 487 LISAFIRTLR 0.080 275YCWEEYRVLR 0.080 438 SVFNLQIYGV 0.080 697 KKNEAPPDNK 0.060 392SNISSPGCEK 0.060 571 CVSAKNAFML 0.060 259 LVLILGVLGV 0.060  49IVVGIVAWLY 0.060 240 LVLSLLFILL 0.060 317 AVLEAILLLM 0.060 362FVLLLICIAY 0.060 433 GLIQRSVFNL 0.054 449 GLFWTLNWVL 0.048 493RTLRYHTGSL 0.045 518 ILEYIDHKLR 0.040 252 LVAGPLVLVL 0.040 618FSGRIPGLGK 0.040 688 SKSLLKILGK 0.040 606 VVGGVGVLSF 0.040 541FKCCLWCLEK 0.040 657 SVFGMCVDTL 0.040 360 VTFVLLLICI 0.040 233LVALGVALVL 0.040 331 RQRIRIAIAL 0.036 589 VVLDKVTDLL 0.030 157TVITSLQQEL 0.030 463 CVLAGAFASF 0.030 588 VVVLDKVTDL 0.030 700EAPPDNKKRK 0.030 314 IVLAVLEAIL 0.030 456 WVLALGQCVL 0.030 257 LVLVLIGVL0.030  34 DVICCVLFLL 0.027 611 GVLSFFFFSG 0.027  59 GDPRQVLYPR 0.024 220KIFEDFAQSW 0.024 654 GFFSVFGMCV 0.024 548 LEKFIKFLNR 0.024 467GAFASFYWAF 0.024 674 LERNNGSLDR 0.024 347 AVGQMMSTMF 0.020 566YGKNFCVSAK 0.020 353 STMFYPLVTF 0.020 585 IVRVVVLDKV 0.020 701APPDNKKRKK 0.020 304 SVQETWLAAL 0.020 380 ATSGQPQYVL 0.020 662CVDTLFLCFL 0.020 414 LVNSSCPGLM 0.020  19 YDPSFRGPIK 0.020 116CVSSCPEDPW 0.020 186 NVTPPALPGI 0.020 642 MTSILGAYVI 0.020 512VQIARVILEY 0.018 478 KPQDIPTFPL 0.018  47 GYIVVGIVAW 0.018 461GQCVLAGAFA 0.018 239 ALVLSLLFIL 0.018   4 KQRDEDDEAY 0.018 603KLLVVGGVGV 0.018 553 KFLNRNAYIM 0.018 163 QQELCPSFLL 0.018 267GVLAYGIYYC 0.018

TABLE XV-V3-HLA-A1101-10 mers-24P4C12 Each peptide is a portion of SEQID NO: 7; each start position is specified, the length of peptide is 10amino acids, and the end postion for each peptide is the start positionplus nine. Start Subsequence Score  9 NITPPALPGI 0.004  5 FPWTNITPPA0.004  3 RCFPWTNITP 0.002  7 WTNITPPALP 0.001 10 ITPPALPGIT 0.001  4CFPWTNITPP 0.000  1 LGRCFPWTNI 0.000  8 TNITPPALPG 0.000  2 GRCFPWTNIT0.000  6 PWTNITPPAL 0.000

TABLE XV-V5-HLA-A1101-10 mers-24P4C12 Each peptide is a portion of SEQID NO: 11; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score  6 ILLLVLIFLR 0.360  1 AVLEAILLLV0.060  9 LVLIFLRQRI 0.030  8 LLVLIFLRQR 0.012 10 VLIFLRQRIR 0.012  5AILLLVLIFL 0.012  2 VLEAILLLVL 0.008  4 EAILLLVLIF 0.002  7 LLLVLIFLRQ0.001  3 LEAILLLVLI 0.001

TABLE XV-V6-HLA-A1101-10 mers-24P4C12 Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score  2 GYSSKGLIPR 0.480  7 GLIPRSVFNL0.054  9 IPRSVFNLQI 0.004  8 LIPRSVFNLQ 0.000  5 SKGLIPRSVF 0.000  6KGLIPRSVFN 0.000  1 QGYSSKGLIP 0.000 10 PRSVFNLQIY 0.000  4 SSKGLIPRSV0.000  3 YSSKGLIPRS 0.000

TABLE XV-V7-HLA-A1101-10 mers-24P4C12 Each peptide is a portion of SEQID NO: 15; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 9 AVGQMMSTMF 0.020 5 WILVAVGQMM 0.0067 LVAVGQMMST 0.004 8 VAVGQMMSTM 0.003 6 ILVAVGQMMS 0.001 3 WYWILVAVGQ0.001 1 QSWYWILVAV 0.000 4 YWILVAVGQM 0.000 2 SWYWILVAVG 0.000

TABLE XV-V8-HLA-A1101-10 mers-24P4C12 Each peptide is a portion of SEQID NO: 17; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 18 HVFQTSILGA 0.080  1 LNYYWLPIMR0.032 21 QTSILGAYVI 0.020 20 FQTSILGAYV 0.006 11 NPITPTGHVF 0.003 13ITPTGHVFQT 0.003 19 VFQTSILGAY 0.002  2 NYYWLPIMRN 0.002 10 RNPITPTGHV0.001 15 PTGHVFQTSI 0.001  6 LPIMRNPITP 0.001  8 IMRNPITPTG 0.000  5WLPIMRNPIT 0.000  4 YWLPIMRNPI 0.000  9 MRNPITPTGH 0.000 14 TPTGHVFQTS0.000 16 TGHVFQTSIL 0.000 17 GHVFQTSILG 0.000  7 PIMRNPITPT 0.000  3YYWLPIMRNP 0.000 12 PITPTGHVFQ 0.000

TABLE XV-V9-HLA-A1101-10 mers-24P4C12 Each peptide is a portion of SEQID NO: 19; each start position is specified, the length of peptide is 10amine acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 13 TQPATLGYVL 0.012  7 ALYPLPTQPA0.008 11 LPTQPATLGY 0.004  9 YPLPTQPATL 0.003 16 ATLGYVLWAS 0.003 14QPATLGYVLW 0.002 19 GYVLWASNIS 0.002  1 AYWAMTALYP 0.002 12 PTQPATLGYV0.001  5 MTALYPLPTQ 0.001  4 AMTALYPLPT 0.001  8 LYPLPTQPAT 0.000 18LGYVLWASNI 0.000 15 PATLGYVLWA 0.000 17 TLGYVLWASN 0.000  3 WAMTALYPLP0.000  2 YWAMTALYPL 0.000  6 TALYPLPTQP 0.000 10 PLPTQPATLG 0.000

TABLE XVI-V1-HLA-A24-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptides is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 356 FYPLVTFVL 420.000  57 LYGDPRQVL288.000 496 RYHTGSLAF 200.000 648 AYVIASGFF 150.000  87 LYFNIFSCI 84.000386 QYVLWASNI 75.000  88 YFNIFSCIL 30.000 666 LFLCFLEDL 30.000 450LFWTLNWVL 24.000 503 AFGALILTL 24.000  84 PYLLYFNIF 21.600 540 CFKCCLWCL20.000 684 PYYMSKSLL 20.000 617 FFSGRIPGL 20.000 658 VFGMCVDTL 20.000553 KFLNRNAYI 15.000 251 RLVAGPLVL 12.000 583 RNIVRVVVL 12.000 484TFPLISAFI 10.500  47 GYIVVGIVA 10.500 301 AYQSVQETW 10.500 468 AFASFYWAF10.000 139 VFYTKNRNF 10.000 518 ILEYIDHKL 9.240 361 TFVLLLICI 9.000 577AFMLLMRNI 9.000 446 GVLGLFWTL 8.640 258 VLVLILGVL 8.400  49 IVVGIVAWL8.400 154 WNMTVITSL 8.400 311 AALIVLAVL 8.400 261 LILGVLGVL 8.400 440FNLQIYGVL 8.400 234 VALGVALVL 8.400 683 RPYYMSKSL 8.000 333 RIRIAIALL8.000 596 DLLLFFGKL 7.920  65 LYPRNSTGA 7.500 328 IFLRQRIRI 7.500 317AVLEAILLL 7.200 255 GPLVLVLIL 7.200  38 CVLFLLFIL 7.200 240 LVLSLLFIL7.200 232 ILVALGVAL 7.200 589 VVLDKVTDL 7.200 170 FLLPSAPAL 7.200 357YPLVTFVLL 7.200 236 LGVALVLSL 7.200 621 RIPGLGKDF 7.200 158 VITSLQQEL6.336 305 VQETWLAAL 6.000  15 KPVKYDPSF 6.000 547 CLEKFIKFL 6.000 597LLLFFGKLL 6.000 565 IYGKNFCVS 6.000  34 DVICCVLFL 6.000 308 TWLAALIVL6.000 184 WTNVTPPAL 6.000 316 LAVLEAILL 6.000 200 TIQQGISGL 6.000 635NYYWLPIMT 6.000 140 FYTKNRNFC 6.000 673 DLERNNGSL 6.000 442 LQIYGVLGL6.000 414 LVNSSCPGL 6.000 444 IYGVLGLFW 6.000 452 WTLNWVLAL 6.000 242LSLLFILLL 6.000 605 LVVGGVGVL 6.000 638 WLPIMTSIL 6.000 511 LVQIARVIL6.000 163 QQELCPSFL 6.000 291 SQLGFTTNL 6.000 434 LIQRSVFNL 6.000 432KGLIQRSVF 6.000 225 FAQSWYWIL 6.000 322 ILLLMLIFL 6.000 593 KVTDLLLFF5.760 241 VLSLLFILL 5.760 253 VAGPLVLVL 5.760 237 GVALVLSLL 5.600 228SWYWILVAL 5.600 249 LLRLVAGPL 5.600  35 VICCVLFLL 5.600  32 CTDVICCVL5.600 590 VLDKVTDLL 5.600 217 ISVKIFEDF 5.040 224 DFAQSWYWI 5.000 614SFFFFSGRI 5.000 274 YYCWEEYRV 5.000 636 YYWLPIMTS 5.000 370 AYWAMTALY5.000 573 SAKNAFMLL 4.800 351 MMSTMFYPL 4.800 315 VLAVLEAIL 4.800 100IISVAENGL 4.800 204 GISGLIDSL 4.800 687 MSKSLLKIL 4.800 244 LLFILLLRL4.800 499 TGSLAFGAL 4.800

TABLE XVI-V3-HLA-A24-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 7; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 6 WTNITPPAL 6.000 9 ITPPALPGI 1.8002 RCFPWTNIT 0.288 1 GRCFPWTNI 0.100 3 CFPWTNITP 0.075 7 TNITPPALP 0.0155 PWTNITPPA 0.014 8 NITPPALPG 0.012 4 FPWTNITPP 0.010

TABLE XVI-V5-HLA-A24-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 11; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 5 ILLLVLIFL 8.400 4 AILLLVLIF 3.6009 VLIFLRQRI 2.160 3 EAILLLVLI 1.800 2 LEAILLLVL 0.480 1 VLEAILLLV 0.2107 LLVLIFLRQ 0.025 6 LLLVLIFLR 0.018 8 LVLIFLRQR 0.015

TABLE XVI-V6-HLA-A24-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 5 KGLIPRSVF 6.000 7 LIPRSVFNL 6.0001 GYSSKGLIP 0.500 6 GLIPRSVFN 0.180 3 SSKGLIPRS 0.120 8 IPRSVFNLQ 0.0204 SKGLIPRSV 0.014 2 YSSKGLIPR 0.010 9 PRSVFNLQI 0.010

TABLE XVI-V7-HLA-A24-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 15; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 5 ILVAVGQMM 1.260 4 WILVAVGWM 0.7502 WYWILVAVG 0.600 8 AVGQMMSTM 0.500 7 VAVGQMMST 0.150 1 SWYWILVAV 0.1406 LVAVGQMMS 0.100 3 YWILVAVGQ 0.021

TABLE XVI V8-HLA-A24-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 17; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score  2 YYWLPIMRN 5.000  4 WLPIMRNPI1.800 15 TGHVFQTSI 1.000 18 VFQTSILGA 0.750 16 GHVFQTSIL 0.600  1NYYWLPIMR 0.600 11 PITPTGHVF 0.240 10 NPITPTGHV 0.150  5 LPIMRNPIT 0.15019 FQTSILGAY 0.140 20 QTSILGAYV 0.120 13 TPTGHVFQT 0.100  7 IMRNPITPT0.100  9 RNPITPTGH 0.030  3 YWLPIMRNP 0.025 14 PTGHVFQTS 0.020 12ITPTGHVFQ 0.015 17 HVFQTSILG 0.010  8 MRNPITPTG 0.003  6 PIMRNPITP 0.002

TABLE XVI-V9-HLA-A24-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 19; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 18 GYVLWASNI 75.000  7 LYPLPTQPA9.000  2 WAMTALYPL 6.000 13 QPATLGYVL 4.800  9 PLPTQPATL 0.600  8YPLPTQPAT 0.180 15 ATLGYVLWA 0.150 12 TQPATLGYV 0.150 16 TLGYVLWAS 0.14017 LGYVLWASN 0.120  4 MTALYPLPT 0.100 11 PTQPATLGY 0.018  5 TALYPLPTQ0.015  6 ALYPLPTQP 0.014  3 AMTALYPLP 0.012 10 LPTQPATLG 0.010 14PATLGYVLW 0.010  1 YWAMTALYP 0.010

TABLE XVII-V1-HLA-A24-10 mers-24P4C12 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 356 FYPLVTFVLL 360.000 301 AYQSVQETWL300.000  87 LYFNIFSCIL 200.000 140 FYTKNRNFCL 200.000 274 YYCWEEYRVL200.000 370 AYWAMTALYL 200.000  18 KYDPSFRGPI 120.000 685 YYMSKSLLKI82.500 636 YYWLPIMTSI 70.000 439 VFNLQIYGVL 42.000 355 MFYPLVTFVL 33.600169 SFLLPSAPAL 30.000 425 VFQGYSSKGL 30.000 616 FFFSGRIPGL 20.000 224DFAQSWYWIL 20.000 478 KPQDIPTFPL 14.400 131 EFSQTVGEVF 14.000 658VFGMCVDTLF 14.000 569 NFCVSAKNAF 12.000 630 KSPHLNYYWL 12.000 493RTLRYHTGSL 12.000 331 RQRIRIAIAL 11.200 517 VILEYIDHKL 11.088  40LFLLFILGYI 10.500 589 VVLDKVTDLL 10.080 157 TVITSLQQEL 9.504 520EYIDHKLRGV 9.000 386 QYVLWASNIS 9.000 445 YGVLGLFWTL 8.640 240LVLSLLFILL 8.640 248 LLLRLVAGPL 8.400 257 LVLVLILGVL 8.400  48YIVVGIVAWL 8.400 260 VLILGVLGVL 8.400 236 LGVALVLSLL 8.400  34DVICCVLFLL 8.400 683 RPYYMSKSLL 8.000 648 AYVIASGFFS 7.500  47GYIVVGIVAW 7.500  65 LYPRNSTGAY 7.500 553 KFLNRNAYIM 7.500 254AGPLVLVLIL 7.200 304 SVQETWLAAL 7.200 231 WILVALGVAL 7.200 637YWLPIMTSIL 7.200 162 LQQELCPSFL 7.200 239 ALVLSLLFIL 7.200 318VLEAILLLML 7.200 314 IVLAVLEAIL 7.200  37 CCVLFLLFIL 7.200 546WCLEKFIKFL 7.200 350 QMMSTMFYPL 7.200  99 NIISVAENGL 7.200 203QGISGLIDSL 7.200 243 SLLFILLLRL 7.200 229 WYWILVALGV 7.000  31SCTDVICCVL 6.720 441 NLQIYGVLGL 6.000 357 YPLVTFVLLL 6.000 604LLVVGGVGVL 6.000 510 TLVQIARVIL 6.000 596 DLLLFFGKLL 6.000 536CIMCCFKCCL 6.000 588 VVVLDKVTDL 6.000 433 GLIQRSVFNL 6.000 659FGMCVDTLFL 6.000 456 WVLALGQCVL 6.000 413 HLVNSSCPGL 6.000 290ISQLGFTTNL 6.000 321 AILLLMLIFL 6.000 316 LAVLEAILLL 6.000  57LYGDPRQVLY 6.000  91 IFSCILSSNI 6.000  77 MGENKDKPYL 6.000 163QQELCPSFLL 6.000 199 TTIQQGISGL 6.000 500 GSLAFGALIL 6.000  83KPYLLYFNIF 5.760 310 LAALIVLAVL 5.600 233 LVALGVALVL 5.600 227QSWYWILVAL 5.600 661 MCVDTLFLCF 5.184 565 IYGKNFCVSA 5.000 279EYRVLRDKGA 5.000 635 NYYWLPIMTS 5.000 273 IYYCWEEYRV 5.000 444IYGVLGLFWT 5.000 686 YMSKSLLKIL 4.800  56 WLYGDPRQVL 4.800 235ALGVALVLSL 4.800 252 LVAGPLVLVL 4.800 449 GLFWTLNWVL 4.800 502LAFGALILTL 4.800 625 LGKDFKSPHL 4.800 498 HTGSLAFGAL 4.800 572VSAKNAFMLL 4.800 542 KCCLWCLEKF 4.400 442 LQIYGVLGLF 4.200 368CIAYWAMTAL 4.000 241 VLSLLFILLL 4.000

TABLE XVII-V3-HLA-A24-10 mers-24P4C12 Each peptide is a portion of SEQID NO: 7; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score  9 NITPPALPGI 1.200  1 LGRCFPWTNI1.000  6 PWTNITPPAL 0.400 10 ITPPALPGIT 0.216  5 FPWTNITPPA 0.140  4CFPWTNITPP 0.075  3 RCFPWTNITP 0.024  8 TNITPPALPG 0.015  7 WTNITPPALP0.015  2 GRCFPWTNIT 0.012

TABLE XVII-V5-HLA-A24-10 mers-24P4C12 Each peptide is a portion of SEQID NO: 11; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score  5 AILLLVLIFL 8.400  2 VLEAILLLVL7.200  4 EAILLLVLIF 3.600  9 LVLIFLRQRI 2.160  1 AVLEAILLLV 0.252  3LEAILLLVLI 0.120  7 LLLVLIFLRQ 0.025  6 ILLLVLIFLR 0.018 10 VLIFLRQRIR0.015  8 LLVLIFLRQR 0.015

TABLE XVII-V6 HLA-A24-10mers-24P4C12 Each peptide is a portion of SEQ IDNO: 13; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 7 GLIPRSVFNL 7.200 9 IPRSVFNLQI 1.0002 GYSSKGLIPR 0.500 6 KGLIPRSVFN 0.300 5 SKGLIPRSVF 0.200 4 SSKGLIPRSV0.140 3 YSSKGLIPRS 0.120 8 LIPRSVFNLQ 0.030 1 QGYSSKGLIP 0.010 10PRSVFNLQIY 0.001

TABLE XVII-V7 HLA-A24-10mers-24P4C12 Each peptide is a portion of SEQ IDNO: 15; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 9 AVGQMMSTMF 2.000 5 WILVAVGQMM 1.2604 YWILVAVGQM 0.750 8 VAVGQMMSTM 0.750 3 WYWILVAVGQ 0.700 6 ILVAVGQMMS0.150 1 QSWYWILVAV 0.140 7 LVAVGQMMST 0.100 2 SWYWILVAVG 0.012

TABLE XVII-V8 HLA-A24-10mers-24P4C12 Each peptide is a portion of SEQ IDNO: 17; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 2 NYYWLPIMRN 5.000 16 TGHVFQTSIL4.000 11 NPITPTGHVF 3.000 4 YWLPIMRNPI 2.160 19 VFQTSILGAY 1.050 21QTSILGAYVI 1.000 3 YYWLPIMRNP 0.700 10 RNPITPTGHV 0.300 14 TPTGHVFQTS0.202 5 WLPIMRNPIT 0.150 13 ITPTGHVFQT 0.150 20 FQTSILGAYV 0.120 18HVFQTSILGA 0.100 15 PTGHVFQTSI 0.100 6 LPIMRNPITP 0.015 7 PIMRNPITPT0.015 8 IMRNPITPTG 0.014 1 LNYYWLPIMR 0.012 9 MRNPITPTGH 0.002 17GHVFQTSILG 0.002 12 PITPTGHVFQ 0.001

TABLE XVII-V9 HLA-A24-10mers-24P4C12 Each peptide is a portion of SEQ IDNO: 19; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 19 GYVLWASNIS 9.000 8 LYPLPTQPAT7.500 13 TQPATLGYVL 7.200 9 YPLPTQPATL 7.200 2 YWAMTALYPL 4.000 18LGYVLWASNI 1.000 1 AYWAMTALYP 0.500 16 ATLGYVLWAS 0.210 7 ALYPLPTQPA0.144 17 TLGYVLWASN 0.120 4 AMTALYPLPT 0.100 14 QPATLGYVLW 0.100 11LPTQPATLGY 0.100 12 PTQPATLGYV 0.018 6 TALYPLPTQP 0.018 3 WAMTALYPLP0.018 15 PATLGYVLWA 0.010 5 MTALYPLPTQ 0.010 10 PLPTQPATLG 0.002

TABLE XVIII-V1 HLA-B7-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 255 GPLVLVLIL 80.000 631 SPHLNYYWL80.000 357 YPLVTFVLL 80.000 683 RPYYMSKSL 80.000 317 AVLEAILLL 60.000249 LLRLVAGPL 40.000 494 TLRYHTGSL 40.000 333 RIRIAIALL 40.000 311AALLVLAVL 36.000 511 LVQIARVIL 30.000 414 LVNSSCPGL 20.000 38 CVLFLLFIL20.000 49 IVVGIVAWL 20.000 446 GVLGLFWTL 20.000 237 GVALVLSLL 20.000 240LVLSLLFIL 20.000 605 LVVGGVGVL 20.000 34 DVICCVLFL 20.000 589 VVLDKVTDL20.000 347 AVGQMMSTM 15.000 573 SAKNAFMLL 12.000 253 VAGPLVLVL 12.000369 IAYWAMTAL 12.000 225 FAQSWYWIL 12.000 213 NARDISVKI 12.000 514IARVILEYI 12.000 154 WNMTVITSL 12.000 316 LAVLEAILL 12.000 234 VALGVALVL12.000 396 SPGCEKVPI 8.000 83 KPYLLYFNI 8.000 406 TSCNPTAHL 6.000 381TSGQPQYVL 6.000 571 CVSAKNAFM 5.000 261 LILGVLGVL 4.000 315 VLAVLEAIL4.000 291 SQLGFTTNL 4.000 638 WLPIMTSIL 4.000 258 VLVLILGVL 4.000 452WTLNWVLAL 4.000 28 KNRSCTDVI 4.000 241 VLSLLFILL 4.000 236 LGVALVLSL4.000 440 FNLQIYGVL 4.000 184 WTNVTPPAL 4.000 597 LLLFFGKLL 4.000 583RNIVRVVVL 4.000 275 YCWEEYRVL 4.000 170 FLLPSAPAL 4.000 596 DLLLFFGKL4.000 282 VLRDKGASI 4.000 158 VITSLQQEL 4.000 537 IMCCFKCCL 4.000 660GMCVDTLFL 4.000 457 VLALGQCVL 4.000 499 TGSLAFGAL 4.000 66 YPRNSTGAY4.000 141 YTKNRIFCL 4.000 555 LNRNAYIMI 4.000 426 GQGYSSKGL 4.000 244LLFILLLRL 4.000 242 LSLLFILLL 4.000 487 LISAFIRTL 4.000 79 ENKDKPYLL4.000 351 MMSTMFYPL 4.000 442 LQIYGVLGL 4.000 200 TIQQGISGL 4.000 434LIQRSVFNL 4.000 501 SLAFGALIL 4.000 322 ILLLMLIFL 4.000 251 RLVAGPLVL4.000 204 GISGLIDSL 4.000 572 VSAKNAFML 4.000 687 MSKSLLKIL 4.000 100IISVAENGL 4.000 232 ILVALGVAL 4.000 302 YQSVQETWL 4.000 35 VICCVLFLL4.000 25 GPIKNRSCT 3.000 482 IPTFPLISA 3.000 344 ASKAVGQMM 3.000 343EASKAVGQM 3.000 149 LPGVPWNMT 3.000 581 LMRNIVRVV 2.000 152 VPWNMTVIT2.000 531 NPVARCIMC 2.000 188 TPPALPGIT 2.000 112 TPQVCVSSC 2.000 60DPRQVLYPR 2.000 525 KLRGVQNPV 2.000 314 IVLAVLEAI 2.000 167 CPSFLLPSA2.000 151 GVPWNMTVI 2.000 192 LPGITNDTT 2.000 359 LVTFVLLLI 2.000 252LVAGPLVLV 1.500 491 FIRTLRYHT 1.500 530 QNPVARCIM 1.500 239 ALVLSLLFI1.200 305 VQETWLAAL 1.200

TABLE XVIII V3-HLA-B7-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 7; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight Start Subsequence Score 6 WTNITPPAL 4.000 9 ITPPALPGI 0.400 4FPWTNITPP 0.200 2 RCFPWTNIT 0.100 1 GRCFPWTNI 0.060 7 TNITPPALP 0.015 8NITPPALPG 0.015 3 CFPWTNITP 0.001 5 PWTNITPPA 0.001

TABLE XVIII-V5 HLA-B7-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 11; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 5 ILLLVLIFL 4.000 3 EAILLLVLI 1.2009 VLIFLRQRI 0.600 2 LEAILLLVL 0.400 4 AILLLVLIF 0.060 1 VLEAILLLV 0.0608 LVLIFLRQR 0.050 7 LLVLIFLRQ 0.010 6 LLLVLIFLR 0.010

TABLE XVIII-V6 HLA-B7-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 7 LIPRSVFNL 4.000 8 IPRSVFNLQ 2.0005 KGLIPRSVF 0.045 6 GLIPRSVFN 0.020 4 SKGLIPRSV 0.020 3 SSKGLIPRS 0.0202 YSSKGLIPR 0.010 9 PRSVFNLQI 0.004 1 GYSSKGLIP 0.001

TABLE XVIII-V7 HLA-B7-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 15; each start position is specified, the length of is 9 aminoacids, and the end position for each peptide is the start position pluseight. Start Subsequence Score 8 AVGQMMSTM 15.000 5 ILVAVGQMM 1.000 4WILVAVGQM 1.000 7 VAVGQMMST 0.300 6 LVAVGQMMS 0.100 1 SWYWILVAV 0.020 3YWILVAVGQ 0.001 2 WYWILVAVG 0.001

TABLE XVIII-V8 HLA-B7-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 17; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 10 NPITPTGHV 6.000 5 LPIMRNPIT 2.00013 TPTGHVFQT 2.000 7 IMRNPITPT 1.500 4 WLPIMRNPI 0.600 15 TGHVFQTSI0.400 16 GHVFQTSIL 0.400 20 QTSILGAYV 0.200 17 HVFQTSILG 0.050 19FQTSILGAY 0.020 18 VFQTSILGA 0.010 12 ITPTGHVFQ 0.010 9 RNPITPTGH 0.0106 PIMRNPITP 0.003 2 YYWLPIMRN 0.003 11 PITPTGHVF 0.002 14 PTGHVFQTS0.002 3 YWLPIMRNP 0.001 8 MRNPITPTG 0.001 1 NYYWLPIMR 0.001

TABLE XVIII-V9 HLA-B7-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 19; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 13 QPATLGYVL 80.000 2 WAMTALYPL36.000 8 YPLPTQPAT 2.000 9 PLPTQPATL 0.400 10 LPTQPATLG 0.300 15ATLGYVLWA 0.300 12 TQPATLGYV 0.200 4 MTALYPLPT 0.100 5 TALYPLPTQ 0.04518 GYVLWASNI 0.040 3 AMTALYPLP 0.030 6 ALYPLPTQP 0.030 17 LGYVLWASN0.020 16 TLGYVLWAS 0.020 7 LYPLPTQPA 0.015 14 PATLGYVLW 0.006 11PTQPATLGY 0.002 1 YWAMTALYP 0.001

TABLE XIX-V1 HLA-B7-10mers-24P4C12 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 478 KPQDIPTFPL 120.000 683 RPYYMSKSLL80.000 357 YPLVTFVLLL 80.000 331 RQRIRIAIAL 40.000 571 CVSAKNAFML 20.000257 LVLVLILGVL 20.000 456 WVLALGQCVL 20.000 588 VVVLDKVTDL 20.000 157TVITSLQQEL 20.000 240 LVLSLLFILL 20.000 66 YPRNSTGAYC 20.000 314ILAVLEAIL 20.000 589 VVLDKVTDLL 20.000 252 LVAGPLVLVL 20.000 657SVFGMCVDTL 20.000 304 SVQETWLAAL 20.000 34 DVICCVLFLL 20.000 233LVALGVALVL 20.000 380 ATSGQPQYVL 18.000 317 AVLEAILLLM 15.000 321AILLLMLIFL 12.000 502 LAFGALILTL 12.000 239 ALVLSLLFIL 12.000 659FGMCVDTLFL 12.000 254 AGPLVLVLIL 12.000 350 QMMSTMFYPL 12.000 235ALGVALVLSL 12.000 536 CIMCCFKCCL 12.000 316 LAVLEAILLL 12.000 310LAALIVLAVL 12.000 585 IVRVVVLDKV 10.000 56 WLYGDPRQVL 9.000 192LPGITNDTTI 8.000 510 TLVQIARVIL 6.000 662 CVDTLGLCFL 6.000 329FLRQRIRIAI 6.000 405 NTSCNPTAHL 6.000 414 LVNSSCPGLM 5.000 413HLVNSSCPGL 4.000 203 QGISGLIDSL 4.000 368 CIAYWAMTAL 4.000 686YMSKSLLKIL 4.000 99 NIISVAENGL 4.000 665 TLFLCFLEDL 4.000 290 ISQLGFTTNL4.000 441 NLQIYGVLGL 4.000 630 KSPHLNYYWL 4.000 315 VLAVLEAILL 4.000 236LGVALVLSLL 4.000 596 DLLLFFGKLL 4.000 60 DPRQVLYPRN 4.000 243 SLLFILLLRL4.000 37 CCVLFLLFIL 4.000 449 GLFWTLNWVL 4.000 162 LQQELCPSFL 4.000 625LGKDFKSPHL 4.000 227 QSWYWILVAL 4.000 498 HTGSLAFGAL 4.000 48 YIVVGIVAWL4.000 604 LLVVGGVGVL 4.000 149 LPGVPWNMTV 4.000 260 VLILGVLGVL 4.000 493RTLRYHTGSL 4.000 248 LLLRLVAGPL 4.000 231 WILVALGVAL 4.000 500GSLAFGALIL 4.000 546 WCLEKFIKFL 4.000 241 VLSLLGILLL 4.000 539CCFKCCLWCL 4.000 445 YGVLGLFWTL 4.000 307 ETWLAALIVL 4.000 435IQRSVFNLQI 4.000 572 VSAKNAFMLL 4.000 433 GLIQRSVFNL 4.000 517VILEYIDHKL 4.000 199 TTIQQGISGL 4.000 31 SCTDVICCVL 4.000 178 LGRCFPWTNV3.000 343 EASKAVGQMM 3.000 346 KAVGQMMSTM 3.000 581 LMRNIVRVVV 3.000 573SAKNAFMLLM 3.000 652 ASGFFSVFGM 3.000 402 VPINTSCNPT 2.000 182FPWTNVTPPA 2.000 528 GVQNPVARCI 2.000 281 RVLRDKGASI 2.000 186NVTPPALPGI 2.000 143 KNRNFCLPGV 2.000 639 LPIMTSILGA 2.000 249LLRLVAGPLV 2.000 172 LPSAPALGRC 2.000 485 FPLISAFIRT 2.000 264GVLGVLAYGI 2.000 531 NPVARCIMCC 2.000 163 QQELCPSFLL 1.800 529VQNPVARCIM 1.500 576 NAFMLLMRNI 1.200 370 AYWAMTALYL 1.200 318VLEAILLLML 1.200

TABLE XIX-V3 HLA-B7-10mers-24P4C12 Each peptide is a portion of SEQ IDNO: 7; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 1 LGRCFPWTNI 6.000 5 FPWTNITPPA 2.0009 NITPPALPGI 0.400 10 ITPPALPGIT 0.100 6 PWTNITPPAL 0.040 8 TNITPPALPG0.015 7 WTNITPPAIP 0.015 3 RCFPWTNITP 0.010 2 GRCFPWTNIT 0.010 4CFPWTNITPP 0.001

TABLE XIX-V5 HLA-87-10mers-24P4C12 Each peptide is a portion of SEQ IDNO: 11; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 5 AILLLVLIFL 12.000 9 LVLIFLRQRI3.000 1 AVLEAILLLV 3.000 2 VLEAILLLVL 1.200 4 EAILLLVLIF 0.060 3LEAILLLVLI 0.040 7 LLLVLIFLRQ 0.010 6 ILLLVLIFLR 0.010 10 VLIFLRQRIR0.010 8 LLVLIFLRQR 0.010

TABLE XIX-V6 HLA-B7-10mers-24P4C12 Each peptide is a portion of SEQ IDNO: 13; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 9 IPRSVFNLQI 80.000 7 GLIPRSVFNL4.000 4 SSKGLIPRSV 0.200 6 KGLIPRSVFN 0.020 3 YSSKGLIPRS 0.020 8LIPRSVFNLQ 0.010 1 QGYSSKGLIP 0.010 5 SKGLIPRSVF 0.005 2 GYSSKGLIPR0.001 10 PRSVFNLQIY 0.000

TABLE XIX-V7-HLA-B7-10 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 15; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 8 VAVGQMMSTM 3.000 5 WILVAVGQMM 1.0007 LVAVGQMMST 0.500 9 AVGQMMSTMF 0.300 1 QSWYWILVAV 0.200 4 YWILVAVGQM0.100 6 ILVAVGQMMS 0.020 2 SWYWILVAVG 0.001 3 WYWILVAVGQ 0.001

TABLE XIX-V8-HLA-B7-10 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 17; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 16 TGHVFQTSIL 4.000 18 HVFQTSILGA0.500 11 NPITPTGHVF 0.400 21 QTSILGAYVI 0.400 14 TPTGHVFQTS 0.400 10RNPITPTGHV 0.300 20 FQTSILGAYV 0.200  6 LPIMRNPITP 0.200 13 ITPTGHVFQT0.100  8 IMRNPITPTG 0.100  5 WLPIMRNPIT 0.100  4 YWLPIMRNPI 0.060  7PIMRNPITPT 0.045 15 PTGHVFQTSI 0.040  1 LNYYWLPIMR 0.010  2 NYYWLPIMRN0.003 19 VFQTSILGAY 0.002 17 GHVFQTSILG 0.001  3 YYWLPIMRNP 0.001 12PITPTGHVFQ 0.001  9 MRNPITPTGH 0.001

TABLE XIX-V9-HLA-07-10 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 19; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score  9 YPLPTQPATL 80.000 13 TQPATLGYVL4.000  7 ALYPLPTQPA 0.450 11 LPTQPATLGY 0.400 14 QPATLGYVLW 0.400  2YWAMTALYPL 0.400 18 LGYVLWASNI 0.400  4 AMTALYPLPT 0.300  3 WAMTALYPLP0.090 16 ATLGYVLWAS 0.060  6 TALYPLPTQP 0.030 15 PATLGYVLWA 0.030 12PTQPATLGYV 0.020 17 TLGYVLWASN 0.020  5 MTALYPLPTQ 0.015  8 LYPLPTQPAT0.010  1 AYWAMTALYP 0.003 19 GYVLWASNIS 0.002 10 PLPTQPATLG 0.002

TABLE XX-V1-HLA-B35-9 meres-24P4C12 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score  66 YPRNSTGAY 120.000 683 RPYYMSKSL40.000 15 KPVKYDPSF 40.000 344 ASKAVGQMM 30.000 437 RSVFNLQIY 20.000 679GSLDRPYYM 20.000 357 YPLVTFVLL 20.000 255 GPLVLVLIL 20.000 631 SPHLNYYWL20.000  83 KPYLLYFNI 16.000 687 MSKSLLKIL 15.000 396 SPGCEKVPI 12.000 69 NSTGAYCGM 10.000 573 SANKAFMLL 9.000 533 VARCIMCCF 9.000 213NARDISVKI 7.200 465 LAGAFASFY 6.000  11 EAYGKPVKY 6.000 333 RIRIAIALL6.000 343 EASKAVGQM 6.000 489 SAFIRTLRY 6.000  79 ENKDKPYLL 6.000 379LATSGQPQY 6.000 558 NAYIMIAIY 6.000 630 KSPHLNYYW 5.000 381 TSGQPQYVL5.000 217 ISVKIFEDF 5.000 132 FSQTVGEVF 5.000 242 LSLLFILLL 5.000 406TSCNPTAHL 5.000 572 VSAKNAFML 5.000 316 LAVLEAILL 4.500 593 KVTDLLLFF4.000 514 IARVILEYI 3.600 287 GASISQLGF 3.000 238 VALVLSLLF 3.000 311AALIVLAVL 3.000 275 YCWEEYRVL 3.000 253 VAGPLVLVL 3.000 651 IASGFFSVF3.000 647 GAYVIASGF 3.000 225 FAQSWYWIL 3.000 174 SAPALGRCF 3.000 234VALGVALVL 3.000 369 IAYWAMTAL 3.000 141 YTKNRNFCL 3.000 494 TLRYHTGSL3.000 678 NGSLDRPYY 3.000 249 LLRLVAGPL 3.000 117 VSSCPEDPW 2.500 282VLRDKGASI 2.400  28 KNRSCTDVI 2.400 317 AVLEAILLL 2.000 266 LGVLAYGIY2.000 363 VLLLICIAY 2.000 267 GVLAYGIYY 2.000  25 GPIKNRSCT 2.000 415VNSSCPGLM 2.000  50 VVGIVAWLY 2.000 589 VVLDKVTDL 2.000 272 GIYYCWEEY2.000 188 TPPALPGIT 2.000 432 KGLIQRSVF 2.000 152 VPWNMTVIT 2.000 192LPGITNDTT 2.000 531 NPVARCIMC 2.000 583 RNIVRVVVL 2.000 366 LICIAYWAM2.000 546 WCLEKFIKF 2.000 554 FLNRNAYIM 2.000 513 QIARVILEY 2.000  92FSCILSSNI 2.000 530 QNPVARCIM 2.000 133 SQTVGEVFY 2.000 251 RLVAGPLVL2.000 409 NPTAHLVNS 2.000 347 AVGQMMSTM 2.000 634 LNYYWLPIM 2.000 621RIPGLGKDF 2.000 643 TSILGAYVI 2.000 482 IPTFPLISA 2.000 110 CPTPQVCVS2.000 641 IMTSILGAY 2.000 677 NNGSLDRPY 2.000 421 GLMCVFQGY 2.000 162LQQELCPSF 2.000 149 LPGVPWNMT 2.000 263 LGVLGVLAY 2.000 167 CPSFLLPSA2.000 148 CLPGVPWNM 2.000 571 CVSAKNAFM 2.000 112 TPQVCVSSC 2.000 384QPQYVLWAS 2.000 500 GSLAFGALI 2.000 349 GQMMSTMFY 2.000 653 SGFFSVFGM2.000   4 KQRDEDDEA 1.800 660 GMCVDTLFL 1.500  30 RSCTDVICC 1.500 430SSKGLIQRS 1.500

TABLE XX-V3-HLA-B35-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 7; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 6 WTNITPPAL 1.000 9 ITPPALPGI 0.4004 FPWTNITPP 0.200 2 RCFPWTNIT 0.200 1 GRCFPWTNI 0.040 7 TNITPPALP 0.0108 NITPPALPG 0.010 3 CFPWTNITP 0.001 5 PWTNITPPA 0.001

TABLE XX-V5-HLA-B35-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 3 EAILLLVLI 1.200 5 ILLLVLIFL 1.0004 AILLLVLIF 1.000 9 VLIFLRQRI 0.400 2 LEAILLLVL 0.100 1 VLEAILLLV 0.0606 LLLVLIFLR 0.010 7 LLVLIFLRQ 0.010 8 LVLIFLRQR 0.010

TABLE XX-V6-HLA-B35-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 5 KGLIPRSVF 2.000 3 SSKGLIPRS 1.5007 LIPRSVFNL 1.000 8 IPRSVFNLQ 0.600 6 GLIPRSVFN 0.100 2 YSSKGLIPR 0.0504 SKGLIPRSV 0.020 9 PRSVFNLQI 0.004 1 GYSSKGLIP 0.001

TABLE XX-V7-HLA-B35-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 15; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 8 AVGQMMSTM 2.000 5 ILVAVGQMM 2.0004 WILVAVGQM 2.000 7 VAVGQMMST 0.300 6 LVAVGQMMS 0.100 1 SWYWILVAV 0.0203 YWILVAVGQ 0.001 2 WYWILVAVG 0.001

TABLE XX-V8-HLA-B35-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 17; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 10 NPITPTGHV 4.000 13 TPTGHVFQT2.000 19 FQTSILGAY 2.000  5 LPIMRNPIT 2.000 15 TGHVFQTSI 0.400  4WLPIMRNPI 0.400  7 IMRNPITPT 0.300 20 QTSILGAYV 0.200 11 PITPTGHVF 0.10016 GHVFQTSIL 0.100  9 RNPITPTGH 0.020  2 YYWLPIMRN 0.010 18 VFQTSILGA0.010 14 PTGHVFQTS 0.010 17 HVFQTSILG 0.010 12 ITPTGHVFQ 0.010  6PIMRNPITP 0.001  3 YWLPIMRNP 0.001 8 MRNPITPTG 0.001 1 NYYWLPIMR 0.001

TABLE XX V9-HLA-B35-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 19; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Start Subsequence Score 13 QPATLGYVL 20.000  2 WAMTALYPL3.000  8 YPLPTQPAT 2.000 11 PTQPATLGY 0.200 12 TQPATLGYV 0.200 10LPTQPATLG 0.200 14 PATLGYVLW 0.150 15 ATLGYVLWA 0.100  4 MTALYPLPT 0.10016 TLGYVLWAS 0.100 17 LGYVLWASN 0.100  9 PLPTQPATL 0.100 18 GYVLWASNI0.040  5 TALYPLPTQ 0.030  7 LYPLPTQPA 0.010  3 AMTALYPLP 0.010  6ALYPLPTQP 0.010  1 YWAMTALYP 0.001

TABLE XXI-V1-HLA-B35-10 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 478 KPQDIPTFPL 80.000  83 KPYLLYFNIF40.000 683 RPYYMSKSLL 40.000   4 KQRDEDDEAY 36.000 123 DPWTVGKNEF 20.000482 IPTFPLISAF 20.000 357 YPLVTFVLLL 20.000 213 NARDISVKIF 18.000 573DAKNAFMLLM 18.000 346 KAVGQMMSTM 12.000  79 ENKDKPYLLY 12.000 652ASGFFSVFGM 10.000 488 ISAFIRTLRY 10.000 132 FSQTVGEVFY 10.000 175APALGRCFPW 10.000 630 KSPHLNYYWL 10.000 192 LPGITNDTTI 8.000 551FIKFLNRNAY 6.000 625 LGKDFKSPHL 6.000 331 RQRIRIAIAL 6.000 343EASKAVGQMM 6.000  60 DPRQVLYPRN 6.000  66 YPRNSTGAYC 6.000 369IAYWAMTALY 6.000 572 VSAKNAFMLL 5.000 227 QSWYWILVAL 5.000 500GSLAFGALIL 5.000 417 SSCPGLMCVF 5.000 290 ISQLGFTTNL 5.000  76GMGENKDKPY 4.000  68 RNSTGAYCGM 4.000 317 AVLEAILLLM 4.000 557RNAYIMIAIY 4.000 149 LPGVPWNMTV 4.000 676 RNNGSLDRPY 4.000 310LAALIVLAVL 3.000 316 LAVLEAILLL 3.000 320 EAILLLMLIF 3.000 467GAFASFYWAF 3.000 395 SSPGCEKVPI 3.000 647 GAYVIASGFF 3.000 677NNGSLDRPYY 3.000 502 LAFGALILTL 3.000 430 SSKGLIQRSV 3.000 381TSGQPQYVLW 2.500 362 FVLLLICIAY 2.000  39 VLFLLFILGY 2.000 188TPPALPGITN 2.000 152 VPWNMTVITS 2.000 348 VGQMMSTMFY 2.000  31SCTDVICCVL 2.000 384 QPQYVLWASN 2.000 409 NPTAHLVNSS 2.000 613LSFFFFSGRI 2.000 220 KIFEDFAQSW 2.000 110 CPTPQVCVSS 2.000 546WCLEKFIKFL 2.000 271 YGIYYCWEEY 2.000  30 RSCTDVICCV 2.000 172LPSAPALGRC 2.000 162 LQQELCPSFL 2.000 396 SPGCEKVPIN 2.000 266LGVLAYGIYY 2.000 402 VPINTSCNPT 2.000 378 YLATSGQPQY 2.000 365LLICIAYWAM 2.000 293 LGFTTNLSAY 2.000 262 ILGVLGVLAY 2.000 286KGASISQLGF 2.000 529 VQNPVARCIM 2.000 678 NGSLDRPYYM 2.000  49IVVGIVAWLY 2.000 147 FCLPGVPWNM 2.000 265 VLGVLAYGIY 2.000 304SVQETWLAAL 2.000 464 VLAGAFASFY 2.000  20 DPSFRGPIKN 2.000 661MCVDTLFLCF 2.000  92 FSCILSSNII 2.000 512 VQIARVILEY 2.000 182FPWTNVTPPA 2.000 639 LPIMTSILGA 2.000 570 FCVSAKNAFM 2.000 493RTLRYHTGSL 2.000 633 HLNYYWLPIM 2.000 531 NPVARCIMCC 2.000 622IPGLGKDFKS 2.000 485 FPLISAFIRT 2.000 542 KSSLWCLEKF 2.000 589VVLDKVTDLL 2.000 517 VILEYIDHKL 2.000 414 LVNSSCPGLM 2.000 344ASKAVGQMMS 1.500 465 LAGAFASFYW 1.500 300 SAYQSVQETW 1.500 659FGMCVDTLFL 1.500 315 VLAVLEAILL 1.500 118 SSCPEDPWTV 1.500 576NAFMLLMRNI 1.200 435 IQRSVFNLQI 1.200

TABLE XXI-V3-HLA-B35-10 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 7; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score  5 FPWTNITPPA 2.000  1 LGRCFPWTNI1.200  9 NITPPALPGI 0.400 10 ITPPALPGIT 0.100  3 RCFPWTNITP 0.020  8TNITPPALPG 0.010  6 PWTNITPPAL 0.010  7 WTNITPPALP 0.010  2 GRCFPWTNIT0.010  4 CFPWTNITPP 0.001

TABLE XXI-V5-HLA-B35-10 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 11; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score  4 EAILLLVLIF 3.000  5 AILLLVLIFL1.000  9 LVLIFLRQRI 0.400 1 AVLEAILLLV 0.400  2 VLEAILLLVL 0.300  3LEAILLLVLI 0.040  6 ILLLVLIFLR 0.010 10 VLIFLRQRIR 0.010  7 LLLVLIFLRQ0.010  8 LLVLIFLRQR 0.010

TABLE XXI-V6-HLA-B35-10 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 13; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score  9 IPRSVFNLQI 24.000  4 SSKGLIPRSV3.000  7 GLIPRSVFNL 1.000  3 YSSKGLIPRS 0.500  6 KGLIPRSVFN 0.200  5SKGLIPRSVF 0.100 10 PRSVFNLQIY 0.020  8 LIPRSVFNLQ 0.010  1 QGYSSKGLIP0.010  2 GYSSKGLIPR 0.001

TABLE XXI-V7-HLA-B35-10 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 15; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 8 VAVGQMMSTM 6.000 5 WILVAVGQMM 2.0009 AVGQMMSTMF 1.000 1 QSWYWILVAV 1.000 4 YWILVAVGQM 0.200 6 ILVAVGQMMS0.100 7 LVAVGQMMST 0.100 2 SWYMILVAVG 0.001 3 WYWILVAVGQ 0.001

TABLE XXI-V8-HLA-B35-10 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 11; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score 11 NPITPTGHVF 20.000 14 TPTGHVFQTS2.000 16 TGHVFQTSIL 1.000 21 QTSILGAYVI 0.400 10 RNPITPTGHV 0.400  6LPIMRNPITP 0.200 20 FQTSILGAYV 0.200 19 VFQTSILGAY 0.200 13 ITPTGHVFQT0.100 18 HVFQTSILGA 0.100  5 WLPIMRNPIT 0.100 15 PTGHVFQTSI 0.040  4YWLPIMRNPI 0.040  8 IMRNPITPTG 0.030  2 NYYWLPIMRN 0.010  7 PIMRNPITPT0.010  1 LNYYWLPIMR 0.010 17 GHVFQTSILG 0.001  3 YYWLPIMRNP 0.001 12PITPTGHVFQ 0.001  9 MRNPITPTGH 0.001

TABLE XXI-V9-HLA-B35-10 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 19; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Start Subsequence Score  2 YWAMTALYPL 1.000 13 TQPATLGYVL1.000  9 YPLPTQPATL 1.000 18 LGYVLWASNI 1.000 14 QPATLGYVLW 0.500 11LPTQPATLGY 0.200 16 ATLGYVLWAS 0.150 19 GYVLWASNIS 0.100  4 AMTALYPLPT0.100  8 LYPLPTQPAT 0.100 17 TLGYVLWASN 0.100  7 ALYPLPTQPA 0.100  3WAMTALYPLP 0.050 12 PTQPATLGYV 0.020  6 TALYPLPTQP 0.010  5 MTALYPLPTQ0.010 15 PATLGYVLWA 0.010  1 AYWAMTALYP 0.010 10 PLPTQPATLG 0.005

Tables XXII-XLIX:

TABLE XXII-V1-HLA-A1-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score  80 NKDKPYLLY 34  58 YGDPRQVLY 33 222FEDFAQSWY 26   5 QRDEDDEAY 25  77 MGENKDKPY 25 263 LGVLGVLAY 24 489SAFIRTLRY 23 513 QIARVILEY 23 628 DFKSPHLNY 22  40 LFLLFILGY 21 267GVLAYGIYY 21 363 VLLLICIAY 21 421 GLMCVFQGY 21  50 VVGIVAWLY 20 318VLEAILLLM 20 629 FKSPHLNYY 20 133 SQATVGEVFY 19 437 RSVFNLQIY 19 662CVDTLFLCF 19  11 EAYGKPVKY 18 370 AYWAMTALY 18  18 KYDPSFRGP 17  32CTDVICCVL 17  66 YPRNSTGAY 17 277 WEEYRVLRD 17 379 LATSGQPQY 17 594VTDLLLFFG 17 165 ELCPSFLLP 16 353 STMFYPLVT 16 398 GCEKVPINT 16 552IKFLNRNAY 16 590 VLDKVTDLL 16 678 NGSLDRPYY 16

TABLE XXII-V3-HLA-A1-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 7; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 8 NITPPALPG 11 9 ITPPALPGI 10 6WTNITPPAL 6 3 CFPWTNITP 5

TABLE XXII-V5-HLA-A1-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 11; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 1 VLEAILLLV 20 7 LLVLIFLRQ 10

TABLE XXII-V6-HLA-A1-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 2 YSSKGLIPR 12 1 GYSSKGLIP 7 3 SSKGLIPRS7 8 IPRSVFNLQ 7 9 PRSVFNLQI 7 6 GLIPRSVFN 5

TABLE XXII-V7-HLA-A1-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 15; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 5 ILVAVGQMM 5 3 YWILVAVGQ 4 7 VAVGQMMST4 6 LVAVGQMMS 3 1 SWYWILVAV 2 2 WYWILVAVG 2

TABLE XXII-V8-HLA-A1-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 17; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 19 FQTSILGAY 16 14 PTGHVFQTS 11 12ITPTGHVFQ 8 18 VFQTSILGA 7 20 QTSILGAYV 7

TABLE XXII-V9-HLA-A1-9 mers-24P4C12 Each peptide is a portion of SEQ IDNO: 19; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 11 PTQPATLGY 31 15 ATLGYVLWA 16

TABLE XXIIl-V1-HLA-A0201-9 mers-24P4C12 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 260 VLILGVLGV 31 244 LLFILLLRL 29 580LLMRNIVRV 29  95 ILSSNIISV 28 204 GISGLIDSL 28 261 LILGVLGVL 28 322ILLLMLIFL 28 506 ALILTLVQI 28 170 FLLPSAPAL 27 252 LVAGPLVLV 27 449GLFWTLNWV 27 487 LISAFIRTL 27 604 LLVVGGVGV 27  45 ILGYIVVGI 26 232ILVALGVAL 26 233 LVALGVALV 26 315 VLAVLEAIL 26 501 SLAFGALIL 26 521YIDHKLRGV 26  42 LLFILGYIV 25 107 GLQCPTPQV 25 200 TIQQGISGL 25 211SLNARDISV 25 239 ALVLSLLFI 25 257 LVLVLILGV 25 258 VLVLILGVL 25 282VLRDKGASI 25 317 AVLEAILLL 25 457 VLALGQCVL 25 598 LLFFGKLLV 25 650VIASGFFSV 25 686 YMSKSLLKI 25  41 FLLFILGYI 24  49 IVVGIVAWL 24 310LAALIVLAV 24 311 AALIVLAVL 24 333 RIRIAIALL 24 434 LIQRSVFNL 24 509LTLVQIARV 24 525 KLRGVQNPV 24 564 AIYGKNFCV 24 581 LMRNIVRVV 24 596DLLLFFGKL 24 605 LVVGGVGVL 24  35 VICCVLFLL 23  56 WLYGDPRQV 23 240LVLSLLFIL 23 251 RLVAGPLVL 23 253 VAGPLVLVL 23 309 WLAALIVLA 23 340LLKEASKAV 23 358 PLVTFVLLL 23 494 TLRYHTGSL 23 518 ILEYIDHKL 23 547CLEKFIKFL 23 589 VVLDKVTDL 23 590 VLDKVTDLL 23 597 LLLFFGKLL 23 100IISVAENGL 22 241 VLSLLFILL 22 248 LLLRLVAGP 22 249 LLRLVAGPL 22 265VLGVLAYGI 22 446 GVLGLFWTL 22 452 WTLNWVLAL 22 578 FMLLMRNIV 22 638WLPIMTSIL 22 660 GMCVDTLFL 22 158 VITSLQQEL 21 187 VTPPALPGI 21 191ALPGITNDT 21 237 GVALVLSLL 21 247 ILLLRLVAG 21 313 LIVLAVLEA 21 314IVLAVLEAI 21 442 LQIYGVLGL 21 507 LILTLVQIA 21 537 IMCCFKCCL 21 599LFFGKLLVV 21 693 KILGKKNEA 21  34 DVICCVLFL 20  38 CVLFLLFIL 20  44FILGYIVVG 20 207 GLIDSLNAR 20 228 SWYWILVAL 20 234 VALGVALVL 20 236LGVALVLSL 20 242 LSLLFILLL 20 319 LEAILLLML 20 326 MLIFLRQRI 20 339ALLKEASKA 20 364 LLLICIAYW 20 417 SSCPGLMCV 20 503 AFGALILTL 20 633HLNYYWLPI 20 644 SILGAYVIA 20 673 DLERNNGSL 20 690 SLLKILGKK 20  48YIVVGIVAW 19 245 LFILLLRLV 19 255 GPLVLVLIL 19 262 ILGVLGVLA 19 268VLAYGIYYC 19 291 SQLGFTTNL 19 318 VLEAILLLM 19 323 LLLMLIFLR 19 329FLRQRIRIA 19 351 MMSTMFYPL 19 365 LLICIAYWA 19 414 LVNSSCPGL 19 464VLAGAFASF 19 544 CLWCLEKFI 19 617 FFSGRIPGL 19 666 LFLCFLEDL 19  86LLYFNIFSC 18 231 WILVALGVA 18 235 ALGVALVLS 18 243 SLLFILLLR 18 336IAIALLKEA 18 355 MFYPLVTFV 18 369 lAYWAMTAL 18 380 ATSGQPQYV 18 394ISSPGCEKV 18 439 VFNLQIYGV 18 459 ALGQCVLAG 18 510 TLVQIARVI 18 511LYQIARVIL 18 514 IARVILEYI 18 517 VILEYIDHK 18 583 RNIVRVVVL 18 602GKLLVVGGV 18 645 ILGAYVIAS 18  46 LGYIVVGIV 17 128 GKNEFSQTV 17 154WNMIVITSL 17 177 ALGRCFPWT 17 184 WTNVTPPAL 17 213 NARDISVKI 17 246FILLLRLVA 17 289 SISQLGFTT 17 300 SAYQSVQET 17 305 VQETWLAAL 17 312ALIVLAVLE 17 325 LMLIFLRQR 17 335 RIAIALLKE 17 354 TMFYPLVTF 17 359LVTFVLLLI 17 453 TLNWVLALG 17 456 WVLALGQCV 17 502 LAFGALILT 17 504FGALILTLV 17 513 QIARVILEY 17 554 FLNRNAYIM 17 560 YIMIAIYGK 17 586VRVVVLDKV 17 642 MTSILGAYV 17 658 VFGMCVDTL 17  31 SCTDVICCV 16  43LFILGYIVV 16  64 VLYPRNSTG 16  90 NIFSCILSS 16 119 SCPEDPWTV 16 144NRNFCLPGV 16 148 CLPGVPWNM 16 161 SLQQELCPS 16 230 YWILVALGV 16 254AGPLVLVLI 16 308 TWLAALIVL 16 316 LAVLEAILL 16 320 EAILLLMLI 16 357YPLVTFVLL 16 362 FVLLLICIA 16 373 AMTALYLAT 16 376 ALYLATSGQ 16 407SCNPTAHLV 16 458 LALGQCVLA 16 637 YWLPIMTSI 16 640 PIMTSILGA 16 52GIVAWLYGD 15 141 YTKNRNFCL 15 225 FAQSWYWIL 15 250 LRLVAGPLV 15 264GYLGYLAYG 15 275 YCWEEYRVL 15 366 LICIAYWAM 15 368 CIAYWAMTA 15 371YWAMTALYL 15 374 MTALYLATS 15 406 TSCNPTAHL 15 433 GLIQRSVFN 15 443QIYGVLGLF 15 491 FIRTLRYHT 15 573 SAKNAFMLL 15 657 SVFGMCVDT 15 663VDTLFLCFL 15

TABLE XXIII-V3-HLA-A0201-9 mers-24P4C12 Each peptide is a portion of SEQID NO: 7; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 9 ITPPALPGI 22 6 WTNITPPAL 17 8NITPPALPG 11 2 RCFPWTNIT 10

TABLE XXIII-V5-HLA-A0201-9 mers-24P4C12 Each peptide is a portion of SEQID NO: 11; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 5 ILLLVLIFL 28 1 VLEAILLLV 25 9VLIFLRQRI 21 2 LEAILLLVL 20 6 LLLVLIFLR 19 3 EAILLLVLI 18 4 AILLLVLIF 187 LLVLIFLRQ 13 8 LVLIFLRQR 13

TABLE XXIII-V6-HLA-A0201-9 mers-24P4C12 Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 2 YSSKGLIPR 12 1 GYSSKGLIP 7 3 SSKGLIPRS7 8 IPRSVFNLQ 7 9 PRSVFNLQI 7 6 GLIPRSVFN 5

TABLE XXIII-V7-HLA-A0201-9 mers-24P4C12 Each peptide is a portion of SEQID NO: 15; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 1 SWYWILVAV 20 4 WILVAVGQM 18 5ILVAVGQMM 16 7 VAVGQMMST 13 8 AVGQMMSTM 12 6 LVAVGQMMS 10

TABLE XXIII-V8-HLA-A0201-9 mers-24P4C12 Each peptide is a portion of SEQID NO: 17; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score  4 WLPIMRNPI 19  7 IMRNPITPT 19 20QTSILGAYV 17 10 NPITPTGHV 15 16 GHVFQTSIL 12 15 TGHVFQTSI 11 18VFQTSILGA 11 12 ITPTGHVFQ 10  5 LPIMRNPIT 9 13 TPTGHVFQT 9

TABLE XXIII V9-HLA-A0201- 9mers-24P4C12 Each peptide is a portion of SEQID NO: 19; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 9 PLPTQPATL 21 2 WAMTALYPL 20 15ATLGYVLWA 20 6 ALYPLPTQP 16 12 TQPATLGYV 14 13 QPATLGYVL 14 16 TLGYVLWAS14 5 TALYPLPTQ 13 4 MTALYPLPT 12 8 YPLPTQPAT 12 3 AMTALYPLP 11

TABLE XXIV V1-HLA-A0203- 9mers-24P4C12 Pos 1234567890 score No ResultsFound.

TABLE XXIV V3-HLA-A0203- 9mers-24P4C12 Pos 1234567890 score No ResultsFound.

TABLE XXIV V5-HLA-A0203- 9mers-24P4C12 Pos 1234567890 score No ResultsFound.

TABLE XXIV V6-HLA-A0203- 9mers-24P4C12 Pos 1234567890 score No ResultsFound.

TABLE XXIV V7-HLA-A0203- 9mers-24P4C12 Pos 1234567890 score No ResultsFound.

TABLE XXIV V8-HLA-A0203- 9mers-24P4C12 Pos 1234567890 score No ResultsFound.

TABLE XXIV V9-HLA-A0203- 9mers-24P4C12 Pos 1234567890 score No ResultsFound.

TABLE XXV V1-HLA-A3- 9mers-24P Each peptide is a portion of SEQ ID NO:3; each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. Pos 123456789 score 585 IVRVVVLDK 29 424 CVFQGYSSK 27 64VLYPRNSTG 26 135 TVGEVFYTK 26 251 RLVAGPLVL 26 506 ALILTLVQI 24 513QIARVILEY 24 603 KLLVVGGVG 24 690 SLLKILGKK 24 267 GVLAYGIYY 23 282VLRDKGASI 23 312 ALIVLAVLE 23 334 IRIAIALLK 23 102 SVAENGLQC 22 232ILVALGVAL 22 247 ILLLRLVAG 22 443 QIYGVLGLF 22 464 VLAGAFASF 22 516RVILEYIDH 22 579 MLLMRNIVR 22 50 VVGIVAWLY 21 212 LNARDISVK 21 281RVLRDKGAS 21 321 AILLLMLIF 21 338 IALLKEASK 21 339 ALLKEASKA 21 376ALYLATSGQ 21 393 NISSPGCEK 21 517 VILEYIDHK 21 593 KVTDLLLFF 21 619SGRIPGLGK 21 621 RIPGLGKDF 21 44 FILGYIVVG 20 56 WLYGDPRQV 20 243SLLFILLLR 20 259 LVLILGVLG 20 347 AVGQMMSTM 20 363 VLLLICIAY 20 463CVLAGAFAS 20 501 SLAFGALIL 20 606 VVGGVGVLS 20 689 KSLLKILGK 20 16PVKYDPSFR 19 170 FLLPSAPAL 19 186 NVTPPALPG 19 207 GLIDSLNAR 19 246FILLLRLVA 19 249 LLRLVAGPL 19 260 VLILGVLGV 19 262 ILGVLGVLA 19 298NLSAYQSVQ 19 317 AVLEAILLL 19 333 RIRIAIALL 19 433 GLIQRSVFN 19 508ILTLVQIAR 19 525 KLRGVQNPV 19 560 YIMIAIYGK 19 588 VVVLDKVTD 19 604LLVVGGVGV 19 605 LVVGGVGVL 19 681 LDRPYYMSK 19 11 EAYGKPVKY 18 49IVVGIVAWL 18 73 AYCGMGENK 18 220 KIFEDFAQS 18 248 LLLRLVAGP 18 261LILGVLGVL 18 264 GVLGVLAYG 18 272 GIYYCWEEY 18 278 EEYRVLRDK 18 314IVLAVLEAI 18 432 KGLIQRSVF 18 441 NLQIYGVLG 18 446 GVLGLFWTL 18 457VLALGQCVL 18 564 AIYGKNFCV 18 587 RVVVLDKVT 18 649 YVIASGFFS 18 10DEAYGKPVK 17 63 QVLYPRNST 17 121 PEDPWTVGK 17 177 ALGRCFPWT 17 211SLNARDISV 17 233 LVALGVALV 17 235 ALGVALVLS 17 239 ALVLSLLFI 17 252LVAGPLVLV 17 309 WLAALIVLA 17 335 RIAIALLKE 17 365 LLICIAYWA 17 368CIAYWAMTA 17 401 KVPINTSCN 17 421 GLMCVFQGY 17 456 WVLALGQCV 17 459ALGQCVLAG 17 510 TLVQIARVI 17 542 KCCLWCLEK 17 562 MIAIYGKNF 17 580LLMRNIVRV 17 583 RNIVRVVVL 17 644 SILGAYVIA 17 657 SVFGMCVDT 17 662CVDTLFLCF 17 26 PIKNRSCTD 16 34 DVICCVLFL 16 45 ILGYIVVGI 16 86LLYFNIFSC 16 157 TVITSLQQE 16 165 ELCPSFLLP 16 237 GVALVLSLL 16 258VLVLILGVL 16 289 SISQLGFTT 16 304 SVQETWLAA 16 323 LLLMLIFLR 16 364LLLICIAYW 16 470 ASFYWAFHK 16 494 TLRYHTGSL 16 511 LVQIARVIL 16 554FLNRNAYIM 16 571 CVSAKNAFM 16 584 NIVRVVVLD 16 673 DLERNNGSL 16 693KILGKKNEA 16 698 KNEAPPDNK 16 20 DPSFRGPIK 15 48 YIVVGIVAW 15 58YGDPRQVLY 15 99 NIISVAENG 15 151 GVPWNMTVI 15 191 ALPGITNDT 15 231WILVALGVA 15 234 VALGVALVL 15 257 LVLVLILGV 15 318 VLEAILLLM 15 322ILLLMLIFL 15 327 LIFLRQRIR 15 329 FLRQRIRIA 15 532 PVARCIMCC 15 589VVLDKVTDL 15 597 LLLFFGKLL 15 598 LLFFGKLLV 15 622 IPGLGKDFK 15 645ILGAYVIAS 15 651 IASGFFSVF 15 680 SLDRPYYMS 15 691 LLKILGKKN 15 7DEDDEAYGK 14 42 LLFILGYIV 14 53 IVAWLYGDP 14 81 KDKPYLLYF 14 95ILSSNIISV 14 148 CLPGVPWNM 14 171 LLPSAPALG 14 244 LLFILLLRL 14 311AALIVLAVL 14 315 VLAVLEAIL 14 324 LLMLIFLRQ 14 326 MLIFLRQRI 14 337AIALLKEAS 14 359 LVTFVLLLI 14 370 AYWAMTALY 14 378 YLATSGQPQ 14 388VLWASNISS 14 453 TLNWVLALG 14 465 LAGAFASFY 14 487 LISAFIRTL 14 496RYHTGSLAF 14 523 DHKLRGVQN 14 527 RGVQNPVAR 14 528 GVQNPVARC 14 534ARCIMCCFK 14 558 NAYIMIAIY 14 567 GKNFCVSAK 14 596 DLLLFFGKL 14 609GVGVLSFFF 14 638 WLPIMTSIL 14 647 GAYVIASGF 14 665 TLFLCFLED 14 685YYMSKSLLK 14 694 ILGKKNEAP 14 699 NEAPPDNKK 14 701 APPDNKKRK 14

TABLE XXV V3-HLA-A3- 9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 7; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 8 NITPPALPG 17

TABLE XXV V5-HLA-A3- 9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 11; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 4 AILLLVLIF 21 8 LVLIFLRQR 20 5ILLLVLIFL 16 6 LLLVLIFLR 16 1 VLEAILLLV 15 7 LLVLIFLRQ 14 9 VLIFLRQRI 14

TABLE XXV V6-HLA-A3- 9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 6 GLIPRSVFN 22 5 KGLIPRSVF 18 7LIPRSVFNL 11

TABLE XXV V7-HLA-A3- 9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 15; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 8 AVGQMMSTM 20 5 ILVAVGQMM 19 6LVAVGQMMS 15 4 WILVAVGQM 14 3 YWILVAVGQ 12 1 SWYWILVAV 10

TABLE XXV V8-HLA-A3- 9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 17; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 11 PITPTGHVF 22 6 PIMRNPITP 16 4WLPIMRNPI 12 9 RNPITPTGH 11 1 NYYWLPIMR 10 17 HVFQTSILG 10

TABLE XXV V9-HLA-A3- 9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 19; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 6 ALYPLPTQP 25 9 PLPTQPATL 18 11PTQPATLGY 12 16 TLGYVLWAS 12

TABLE XXVI-V1 HLA-A26-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 34 DVICCVLFL 35 49 IVVGIVAWL 28 483PTFPLISAF 28 605 LVVGGVGVL 27 593 KVTDLLLFF 26 317 AVLEAILLL 25 592DKVTDLLLF 25 138 EVFYTKNRN 24 240 LVLSLLFIL 24 589 VVLDKVTDL 24 38CVLFLLFIL 23 237 GVALVLSLL 23 11 EAYGKPVKY 22 267 GVLAYGIYY 22 285DKGASISQL 22 452 WTLNWVLAL 22 50 VVGIVAWLY 20 79 ENKDKPYLL 20 157TVITSLQQE 20 263 LGVLGVLAY 20 446 GVLGLFWTL 20 628 DFKSPHLNY 20 641IMTSILGAY 20 662 CVDTLFLCF 20 236 LGVALVLSL 19 258 VLVLILGVL 19 307ETWLAALIV 19 320 EAILLLMLI 19 414 LVNSSCPGL 19 437 RSVFNLQIY 19 513QIARVILEY 19 609 GVGVLSFFF 19 673 DLERNNGSL 19 32 CTDVICCVL 18 198DTTIQQGIS 18 200 TIQQGISGL 18 204 GISGLIDSL 18 244 LLFILLLRL 18 294GFTTNLSAY 18 354 TMFYPLVTF 18 360 VTFVLLLIC 18 400 EKVPINTSC 18 511LVQIARVIL 18 596 DLLLFFGKL 18 102 SVAENGLQC 17 184 WTNVTPPAL 17 216DISVKIFED 17 261 LILGVLGVL 17 358 PLVTFVLLL 17 438 SVFNLQIYG 17 442LQIYGVLGL 17 443 QIYGVLGLF 17 487 LISAFIRTL 17 608 GGVGVLSFF 17 664DTLFLCFLE 17

TABLE XXVI-V3 HLA-A26-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 7; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 6 WTNITPPAL 17 9 ITPPALPGI 13

TABLE XXVI-V5 HLA-A26-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 11; each start position is specified, the length cf peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 3 EAILLLVLI 19 4 AILLLVLIF 18 8LVLIFLRQR 15 2 LEAILLLVL 14 5 ILLLVLIFL 13

TABLE XXVI-V6 HLA-A26-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 7 LIPRSVFNL 16 5 KGLIPRSVF 9

TABLE XXVI-V7 HLA-A26-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 15; each start position is specified, the length of peptide is 9amine acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 8 AVGQMMSTM 12 6 LVAVGQMMS 11 4WILVAVGQM 10 1 SWYWILVAV 8 5 ILVAVGQMM 6 2 WYWILVAVG 5 7 VAVGQMMST 5

TABLE XXVI-V8 HLA-A26-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 17; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 19 FQTSILGAY 20 11 PITPTGHVF 15 17HVFQTSILG 15 16 GHVFQTSIL 13 20 QTSILGAYV 10 14 PTGHVFQTS 9

TABLE XXVI-V9 HLA-A26-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 19; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 11 PTQPATLGY 20 15 ATLGYVLWA 13 2WAMTALYPL 12 13 QPATLGYVL 10 4 MTALYPLPT 9 9 PLPTQPATL 9

TABLE XXVII-V1 HLA-B0702-9mers-24P4C12 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 255 GPLVLVLIL 23 357 YPLVTFVLL 23 683RPYYMSKSL 21 149 LPGVPWNMT 20 396 SPGGEKVPI 20 482 IPTEPLISA 20 631SPHLNYYWL 20 15 KPVKYDPSF 19 152 VPWNMTVIT 19 167 CPSFLLPSA 19 25GPIKNRSCT 18 172 LPSAPALGR 18 83 KPYLLYFNI 17 188 TPPALPGIT 17 192LPGITNDTT 17 57 LYGDPRQVL 16 232 ILVALGYAL 16 253 VAGPLVLVL 16 479PQDIPTFPL 16 503 AFGALILTL 16 49 IVVGIVAWL 15 120 CPEDPWTVG 15 175APALGRCFP 15 189 PPALPGITN 15 234 VALGVALVL 15 251 RLVAGPLVL 15 381TSGQPQYVL 15 406 TSCNPTAHL 15 583 RNIVRVVVL 15 617 FFSGRIPGL 15 20DPSFRGPIK 14 34 DVICCVLFL 14 66 YPRNSTGAY 14 204 GISGLIDSL 14 236LGVALVLSL 14 252 LVAGPLVLV 14 291 SQLGFTTNL 14 311 AALIVLAVL 14 317AVLEAILLL 14 333 RIRIAIALL 14 351 MMSTMFYPL 14 419 CPGLMCVFQ 14 452WTLNWVLAL 14 499 TGSLAFGAL 14 605 LVVGGVGVL 14 660 GMCVDTLFL 14 60DPRQVLYPR 13 100 IISVAENGL 13 110 CPIPQVCVS 13 164 QELQPSELL 13 170FLLPSAPAL 13 182 FPWTNVTPP 13 228 SWYWILVAL 13 241 VLSLLFILL 13 249LLRLVAGPL 13 261 LILGVLGVL 13 302 YQSVQETWL 13 319 LEAILLLML 13 358PLVTFVLLL 13 369 IAYWAMTAL 13 371 YWAMTALYL 13 409 NPTAHLVNS 13 442LQIYGVLGL 13 446 GVLGLFWTL 13 478 KPQDIPTFP 13 487 LISAFIRTL 13 494TLRYHTGSL 13 501 SLAFGALIL 13 511 LVQIARVIL 13 590 VLDKVTDLL 13 622IPGLGKDFK 13 651 IASGFFSVF 13 32 CTDVICCVL 12 78 GENKDKPYL 12 154WNMTVITSL 12 184 WTNVTPPAL 12 242 LSLLFILLL 12 244 LLFILLLRL 12 285DKGASISQL 12 305 VQETWLAAL 12 308 TWLAALIVL 12 315 VLAVLEAIL 12 322ILLLMLIFL 12 356 FYPLVTFVL 12 373 AMTALYLAT 12 380 AISGQPQYV 12 457VLALGQCVL 12 525 KLRGVQNPV 12 547 CLEKEIKEL 12 572 VSAKNAFML 12 589WLDKVTDL1 2 591 LDKVTDLLL 12 626 GKDFKSPHL 12 658 VFGMCVDTL 12 701APPDNKKRK 12 28 KNRSCTDVI 11 45 ILGYIVVGI 11 79 ENKDKPYLL 11 104AENGLQCPT 11 107 GLQCPTPQV 11 109 QCPTPQVCV 11 112 TPQVCVSSC 11 123DPWTVGKNE 11 163 QQELCPSFL 11 169 SFLLPSAPA 11 177 ALGRCFPWT 11 191ALPGITNDT 11 237 GVALVLSLL 11 239 ALVLSLLFI 11 258 VLVLILGVL 11 262ILGVLGVLA 11 275 YCWEEYRVL 11 310 LAALIVLAV 11 332 QRIRIAIAL 11 343EASKAVGQM 11 354 TMFYPLVTF 11 384 QPQYVLWAS 11 414 LVNSSCPGL 11 426FQGYSSKGL 11 434 LIQRSVFNL 11 440 FNLQIYGVL 11 450 LFWTLNWVL 11 464VLAGAFASF 11 518 ILEYIDHKL 11 531 NPVARCIMC 11 537 IMCCFKCCL 11 571CVSAKNAFM 11 573 SAKNAFMLL 11 574 AKNAFMLLM 11 596 DLLLFFGKL 11 597LLLFFGKLL 11 599 LFFGKLLVV 11 638 WLPIMTSIL 11 663 VDTLFLCFL 11 686YMSKSLLKI 11 702 PPDNKKRKK 11

TABLE XXVII-V3 HLA-B0702-9mers-24P4C12 Each peptide is a portion of SEQID NO: 7; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 4 FPWTNITPP 12 6 WTNITPPAL 12 1GRCFPWTNI 10 2 RCFPWTNIT 9 5 PWTNITPPA 9 9 ITPPALPGI 9 8 NITPPALPG 7

TABLE XXVII-V5 HLA-B0702-9mers-24P4C12 Each peptide is a portion of SEQID NO: 11; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 2 LEAILLLVL 14 5 ILLLVLIFL 12 4AILLLVLIF 11 1 VLEAILLLV 9 3 EAILLLVLI 9 9 VLIFLHQHI 7

TABLE XXVII-V6 HLA-B0702-9mers-24P4C12 Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 8 IPRSVFNLQ 14 5 KGUPRSVF 12 7 LIPRSVFNL11 9 PRSVFNLQI 10 4 SKGLIPRSV 7

TABLE XXVII-V7 HLA-B0702-9mers-24P4C12 Each peptide is a portion of SEQID NO: 15; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 1 SWYWILVAV 9 5 ILVAVGQMM 9 8 AVGQMMSTM9 7 VAVGQMMST 8 4 WILVAVGQM 7

TABLE XXVII-V8 HLA-B0702-9mers-24P4C12 Each peptide is a portion of SEQID NO: 17; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 19 FQTSILGAY 20 11 PITPTGHVF 15 17HVFQTSILG 15 16 GHVFQTSIL 13 20 QTSILGAYV 10 14 PTGHVFQTS 9

TABLE XXVII-V9 HLA-B0702-9mers-24P4C12 Each peptide is a portion of SEQID NO: 19; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 13 QPATLGYVL 23 8 YPLPTQPAT 19 10LPTQPATLG 14 15 ATLGYVLWA 13 2 WAMTALYPL 12 7 LYPLPTQPA 11 9 PLPTQPATL11

TABLE XXVIII-V1 HLA-B08-9mers Each peptide is a portion of SEQ ID NO: 3;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. Pos 123456789 score 79 ENKDKPYLL 32 141 YTKNRNFCL 29 282VLRDKGASI 29 573 SAKNAFMLL 26 249 LLRLVAGPL 23 494 TLRYHTGSL 23 26PIKNRSQTD 22 329 FLRQRIRIA 22 589 VVLDKVTDL 22 333 RIRIAIALL 21 583RNIVRVVVL 21 591 LDKVTDLLL 21 626 GKDFKSPHL 21 687 MSKSLLKIL 21 340LLKEASKAV 20 474 WAFHKPQDI 20 523 DHKLRGVQN 20 540 CFKCCLWCL 20 617FFSGRIPGL 20 2 GGKQRDEDD 19 232 ILVALGVAL 19 255 GPLVLVLIL 19 631SPHLNYYWL 19 694 ILGKKNEAP 19 139 VFYTKNRNF 18 170 FLLPSAPAL 18 241VLSLLFILL 18 247 ILLLRLVAG 18 258 VLVLILGVL 18 315 VLAVLEAIL 18 322ILLLMLIFL 18 357 YPLVTFVLL 18 457 VLALGQCVL 18 501 SLAFGALIL 18 514IARVILEYI 18 518 ILEYIDHKL 18 546 WCLEKFIKF 18 547 CLEKFIKFL 18 683RPYYMSKSL 18 11 EAYGKPVKY 17 213 NARDISVKI 17 216 DISVKIFED 17 358PLVTFVLLL 17 533 VARCIMCCF 17 590 VLDKVTDLL 17 596 DLLLFFGKL 17 597LLLFFGKLL 17 673 DLERNNGSL 17 691 LLKILGKKN 17 45 ILGYIVVGI 16 64VLYPRNSTG 16 81 KDKPYLLYF 16 100 IISVAENGL 16 158 VITSLQQEL 16 204GISGLIDSL 16 211 SLNARDISV 16 244 LLFILLLRL 16 251 RLVAGPLVL 16 253VAGPLVLVL 16 338 IALLKEASK 16 369 IAYWAMTAL 16 433 GLIQRSVFN 16 551FIKFLNRNA 16 638 WLPIMTSIL 16 702 PPDNKKRKK 16 35 VICCVLFLL 15 200TIQQGISGL 15 225 FAQSWYWIL 15 234 VALGVALVL 15 316 LAVLEAILL 15 331RQRIRIAIA 15 396 SPGCEKVPI 15 434 LIQRSVFNL 15 487 LISAFIRTL 15 553KFLNRNAYI 15 564 AIYGKNFGV 15 579 MLLMRNIVR 15 693 KILGKKNEA 15

TABLE XXVIII-V3 HLA-B08-9mers-24P4C12 Each peptide is a portion of SEQID NO: 7; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 6 WTNITPPAL 11 4 FPWTNITPP 8 1 GRCFPWTNI7 9 ITPPALPGI 7

TABLE XXVIII-V5 B08-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 11; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 5 ILLLVLIFL 18 3 EAILLLVLI 14 9VLIFLRQRI 13 4 AILLLVLIF 12 2 LEAILLLVL 10 6 LLLVLIFLH 8

TABLE XXVIII-V6 HLA-B08-9mers-24P4C12 Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 6 GLIPRSVFN 16 7 LIPRSVFNL 15 3SSKGLIPRS 13 8 IPRSVFNLQ 13 1 GYSSKGLIP 11 9 PRSVFNLQI 8

TABLE XXVIII-V7 HLA-B08-9mers-24P4C12 Each peptide is a portion of SEQID NO: 15; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 5 ILVAVGQMM 7 4 WILVAVGQM 6 7 VAVGQMMST5 1 SWYWILVAV 4

TABLE XXVIII-V8 HLA-B08-9mers-24P4C12 Each peptide is a portion of SEQID NO: 17; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 5 LPIMRNPIT 15 4 WLPIMRNPI 12 16GHVFQTSIL 11 11 PITPTGHVF 10 7 IMRNPITPT 8 13 TPTGHVFQT 7 15 TGHVFQTSI 7

TABLE XXVIII-V9 HLA-B08-9mers-24P4C12 Each peptide is a portion of SEQID NO: 19; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 9 PLPTQPATL 18 13 QPATLGYVL 16 2WAMTALYPL 14 16 TLGYVLWAS 8 18 GYVLWASNI 8 8 YPLPTQPAT 7

TABLE XXIX-V1 HLA-B1510-9mers-24P4C12 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 275 YCWEEYRVL 18 583 RNIVRVVVL 16 57LYGDPRQVL 15 232 ILVALGVAL 15 253 VAGPLVLVL 15 381 TSGQPQYVL 15 487LISAFIRTL 15 605 LVVGGVGVL 15 49 IVVGIVAWL 14 78 GENKDKPYL 14 100IISVAENGL 14 170 FLLPSAPAL 14 184 WTNVTPPAL 14 200 TIQQGISGL 14 204GISGLIDSL 14 251 RLVAGPLVL 14 357 YFLVTFVLL 14 369 IAYWAMTAL 14 457VLALGQCVL 14 617 FFSGRIPGL 14 32 CIDVICCVL 13 79 ENKDKPYLL 13 228SWYWILVAL 13 234 VALGVALVL 13 255 GPLVLVLIL 13 261 LILGVLGVL 13 302YQSVQETWL 13 308 TWLAALIVL 13 440 FNLQIYGVL 13 446 GVLGLFWTL 13 499TGSLAFGAL 13 511 LVQIARVIL 13 518 ILEYIDHKL 13 537 IMCCFKCCL 13 547CLEKFIKFL 13 572 VSAKNAFML 13 163 QQELCPSFL 12 237 GVALVLSLL 12 244LLFILLLRL 12 258 VLVLILGVL 12 305 VQETWLAAL 12 311 AALIVLAVL 12 315VLAVLEAIL 12 317 AVLEAILLL 12 322 ILLLMLIFL 12 356 FYPLVIFVL 12 371YWAMTALYL 12 406 TSCNPTAHL 12 412 AHLVNSSCP 12 442 LQIYGVLGL 12 450LFWTLNWVL 12 452 WTLNWVLAL 12 476 FHKPQDIPT 12 497 YHTGSLAFG 12 501SLAFGALIL 12 503 AFGALILTL 12 523 DHKLRGVQN 12 589 VVLDKVTDL 12 626GKDFKSPHL 12 651 IASGFFSVF 12 658 VFGMCVDTL 12 660 GMCVDTLFL 12 673DLERNNGSL 12 34 DVICCVLFL 11 88 YFNIFSCIL 11 141 YTKNRNFCL 11 154WNMTVITSL 11 158 VITSLQQEL 11 164 QELCPSFLL 11 236 LGVALVLSL 11 241VLSLLFILL 11 242 LSLLFILLL 11 285 DKGASISQL 11 291 SQLGFTTNL 11 319LEAILLLML 11 332 QRIRIAIAL 11 333 RIRIAIALL 11 351 MMSTMFYPL 11 354TMFYPLVTF 11 358 PLVTFVLLL 11 414 LVNSSCPGL 11 434 LIQRSVFNL 11 479PQDIPTFPL 11 494 TLRYHTGSL 11 590 VLDKVTDLL 11 591 LDKVTDLLL 11 631SPHLNYYWL 11 684 PYYMSKSLL 11 35 VICCVLFLL 10 38 CVLFLLFIL 10 124PWTVGKNEF 10 225 FAQSWYWIL 10 240 LVLSLLFIL 10 249 LLRLVAGPL 10 316LAVLEAILL 10 343 EASKAVGQM 10 418 SCPGLMGVF 10 426 FQGYSSKGL 10 477HKPQDIPTF 10 483 PTFPLISAF 10 540 CFKCCLWCL 10 573 SAKNAFMLL 10 596DLLLFFGKL 10 597 LLLFFGKLL 10 632 PHLNYYWLP 10 638 WLPIMTSIL 10 663VDTLFLCFL 10 666 LFLCFLEDL 10 683 RPYYMSKSL 10 687 MSKSLLKIL 10 33TDVICCVLF 9 36 ICCVLFLLF 9 217 ISVKIFEDF 9 347 AVGQMMSTM 9 432 KGLIQRSVF9 461 GQCVLAGAF 9 607 VGGVGVLSF 9 679 GSLDRPYYM 9 15 KPVKYDPSF 8 81KDKPYLLYF 8 132 FSQTVGEVF 8 139 VFYTKNRNF 8 148 CLPGVPWNM 8 162LQQELCPSF 8 174 SAPALGRCF 8 287 GASISQLGF 8 415 VNSSCPGLM 8 464VLAGAFASF 8 468 AFASFYWAF 8 496 RYHTGSLAF 8 530 QNPVARCIM 8 570FCVSAKNAF 8 608 GGVGVLSFF 8 609 GVGVLSFFF 8 647 GAYVIASGF 8 48 YIVVGIVAW7 69 NSTGAYCGM 7 214 ARDISVKIF 7 238 VALVLSLLF 7 318 VLEAILLLM 7 321AILLLMLIF 7 366 LICIAYWAM 7 443 QIYGVLGLF 7 533 VARCIMCCF 7 546WCLEKFIKF 7 554 FLNRNAYIM 7 562 MIAIYGKNF 7 571 CVSAKNAFM 7 574AKNAFMLLM 7 593 KVTDLLLFF 7 621 RIPGLGKDF 7 634 LNYYWLPIM 7 653SGFFSVFGM 7

TABLE XXIX-V3 HLA-B1510-9mers-24P4C12 Each peptide is a portion of SEQID NO: 7; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 6 WTNITPPAL 13

TABLE XXIX-V5 B1510-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 11; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 2 LEAILLLVL 13 5 ILLLVLIFL 12

TABLE XXIX-V6 B1510-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 7 LIPRSVFNL 11 5 KGLIPRSVF 10 3SSKGLIPRS 5 6 GLIPRSVFN 5

TABLE XXIX-V7 B1510-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 15; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 8 AVGQMMSTM 9 4 WILVAVGQM 8 5 ILVAVGQMM8 1 SWYWILVAV 3 2 WYWILVAVG 3 3 YWILVAVGQ 3 6 LVAVGQMMS 3

TABLE XXIX-V8 B1510-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 17; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 16 GHVFQTSIL 21 11 PITPTGHVF 10 13QPATLGYVL 13 9 PLPTQPATL 12 2 WAMTALYPL 10

TABLE XXIX-V9 B1510-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 19; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 13 QPATLGYVL 13 9 PLPTQPATL 12 2WAMTALYPL 10

TABLE XXX-V1 HLA-B2705-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 334 IRIAIALLK 26 332 QRIRIAIAL 25 675ERNNGSLDR 24 214 ARDISVKIF 23 534 ARCIMCCFK 21 620 GRIPGLGKD 21 5QRDEDDEAY 20 204 GISGLIDSL 20 446 GVLGLFWTL 20 689 KSLLKILGK 20 251RLVAGPLVL 19 424 CVFQGYSSK 19 436 QRSVFNLQI 19 483 PTFPLISAF 19 583RNIVRVVVL 19 608 GGVGVLSFF 19 15 KPVKYDPSF 18 22 SFRGPIKNR 18 179GRCFPWTNV 18 200 TIQQGISGL 18 207 GLIDSLNAR 18 234 VALGVALVL 18 244LLFILLLRL 18 255 GPLVLVLIL 18 291 SQLGFTTNL 18 317 AVLEAILLL 18 330LHQHIRIAI 18 333 RIRIAIALL 18 496 RYHTGSLAF 18 527 RGVQNPVAR 18 647GAYVIASGF 18 668 LCFLEDLER 18 683 RPYYMSKSL 18 690 SLLKILGKK 18 49IVVGIVAWL 17 78 GENKDKPYL 17 154 WNMTVITSL 17 237 GVALVLSLL 17 242LSLLFILLL 17 261 LILGVLGVL 17 287 GASISQLGF 17 311 AALIVLAVL 17 338IALLKEASK 17 354 TMFYPLVTF 17 381 TSGQPQYVL 17 429 YSSKGLIQR 17 477HKPQDIPTF 17 503 AFGALILTL 17 516 RVILEYIDH 17 546 WCLEKFIKF 17 549EKFIKFLNR 17 605 LVVGGVGVL 17 621 RIPGLGKDF 17 11 EAYGKPVKY 16 23FRGPIKNRS 16 137 GEVFYTKNR 16 139 VFYTKNRNF 16 170 FLLPSAPAL 16 283LRDKGASIS 16 285 DKGASISQL 16 321 AILLLMLIF 16 322 ILLLMLIFL 16 323LLLMLIFLR 16 327 LIFLRQRIR 16 432 KGLIQRSVF 16 440 FNLQIYGVL 16 442LQIYGVLGL 16 443 QIYGVLGLF 16 457 VLALGQCVL 16 508 ILTLVQIAR 16 517VILEYIDHK 16 589 VVLDKVTDL 16 617 FFSGRIPGL 16 626 GKDFKSPHL 16 699NEAPPDNKK 16 10 DEAYGKPVK 15 40 LFLLFILGY 15 60 DPRQVLYPR 15 73AYGGMGENK 15 81 KDKPYLLYF 15 124 PWTVGKNEF 15 212 LNARDISVK 15 217ISVKIFEDF 15 228 SWYWILVAL 15 236 LGVALVLSL 15 238 VALVLSLLF 15 243SLLFILLLR 15 253 VAGPLVLVL 15 258 VLVLILGVL 15 308 TWLAALIVL 15 316LAVLEAILL 15 369 IAYWAMTAL 15 461 GQCVLAGAF 15 470 ASFYWAFHK 15 518ILEYIDHKL 15 542 KCCLWCLEK 15 543 CCLWCLEKF 15 547 CLEKFIKFL 15 567GKNFCVSAK 15 579 MLLMRNIVR 15 586 VRVVVLDKV 15 593 KVTDLLLFF 15 596DLLLFFGKL 15 607 VGGVGVLSF 15 609 GVGVLSFFF 15 622 IPGLGKDFK 15 651IASQFFSVF 15 684 PYYMSKSLL 15 698 KNEAPPDNK 15 34 DVICCVLFL 14 38CVLFLLFIL 14 61 PRQVLYPRN 14 75 CGMGENKDK 14 83 KPYLLYFNI 14 84PYLLYFNIF 14 135 TVGEVFYTK 14 148 CLPGVPWNM 14 158 VITSLQQEL 14 162LQQELCPSF 14 164 QELCPSFLL 14 232 ILVALGVAL 14 240 LVLSLLFIL 14 263LGVLGVLAY 14 267 GVLAYGIYY 14 272 GIYYCWEEY 14 278 EEYRVLRDK 14 325LMLIFLRQR 14 379 LATSGQPQY 14 418 SCPGLMCVF 14 434 LIQRSVFNL 14 437RSVFNLQIY 14 450 LFWTLNWVL 14 452 WTLNWVLAL 14 464 VLAGAFASF 14 485EPLISAFIR 14 487 LISAFIRTL 14 488 ISAFIRTLR 14 489 SAFIRTLRY 14 501SLAFGALIL 14 513 QIARVILEY 14 515 ARVILEYID 14 552 IKFLNRNAY 14 556NRNAYIMIA 14 558 NAYIMIAIY 14 560 YIMIAIYGK 14 575 KNAFMLLMR 14 585IVRVVVLDK 14 595 TDLLLFFGK 14 613 LSFFFFSGR 14 643 TSILGAYVI 14 659FGMCVDTLF 14 660 GMCVDTLFL 14 679 GSLDRPYYM 14 700 EAPPDNKKR 14 701APPDNKKRK 14 702 PPDNKKRKK 14 7 DEDDEAYGK 13 36 ICCVLFLLF 13 172LPSAPALGR 13 241 VLSLLFILL 13 249 LLRLVAGPL 13 250 LRLVAGPLV 13 273IYYCWEEYR 13 275 YCWEEYRVL 13 280 YRVLRDKGA 13 294 GFTTNLSAY 13 319LEAILLLML 13 347 AVGQMMSTM 13 348 VGQMMSTMF 13 349 GQMMSTMFY 13 356FYPLVTFVL 13 357 YPLVTFVLL 13 358 PLVTFVLLL 13 363 VLLLICIAY 13 492IRILRYHTG 13 495 LRYHTGSLA 13 506 ALILTLVQI 13 526 LRGVQNPVA 13 545LWCLEKFIK 13 570 FCVSAKNAF 13 572 VSAKNAFML 13 582 MRNIVRVVV 13 590VLDKVTDLL 13 592 DKVTDLLLF 13 610 VGVLSFFFF 13 637 YWLPIMTSI 13 648AYVIASGFF 13 653 SGFFSVFGM 13 666 LFLCFLEDL 13 681 LDRPYYMSK 13 682DRPYYMSKS 13 685 YYMSKSLLK 13 686 YMSKSLLKI 13 29 NRSCTDVIC 12 32GTDVICCVL 12 33 TDVICCVLF 12 35 VICCVLFLL 12 57 LYGDPRQVL 12 58YGDPRQVLY 12 79 ENKDKPYLL 12 80 NKDKPYLLY 12 93 SCILSSNII 12 100IISVAENGL 12 121 PEDPWTVGK 12 132 FSQTVGEVF 12 144 NRNFCLPGV 12 151GVPWNMTVI 12 163 QQELCPSFL 12 190 PALPGITND 12 193 PGITNDTTI 12 239ALVLSLLFI 12 276 CWEEYRVLR 12 302 YQSVQETWL 12 305 VQETWLAAL 12 315VLAVLEAIL 12 320 EAILLLMLI 12 328 IFLRQRIRI 12 343 EASKAVGQM 12 371YWAMTALYL 12 386 QYVLWASNI 12 393 NISSPGCEK 12 406 TSCNPTAHL 12 414LVNSSCPGL 12 421 GLMCVFQGY 12 426 FQGYSSKGL 12 468 AFASFYWAF 12 490AFIRTLRYH 12 500 GSLAFGALI 12 510 TLVQIARVI 12 519 LEYIDHKLR 12 537IMCCFKCCL 12 540 CFKCCLWCL 12 553 KFLNRNAYI 12 557 RNAYIMIAI 12 562MIAIYGKNF 12 591 LDKVTDLLL 12 597 LLLFFGKLL 12 614 SFFFFSGRI 12 619SGRIPGLGK 12 628 DFKSPHLNY 12 631 SPHLNYYWL 12 634 LNYYWLPIM 12 658VFGMCVDTL 12 662 CVDTLFLCF 12 663 VDTLFLCFL 12 673 DLERNNGSL 12 687MSKSLLKIL 12

TABLE XXX-V3 HLA-B2705-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 7; each start position is specified, the length of peptide is 9amino acids, and the end positien for each peptide is the start positionplus eight. Pos 123456789 score 1 GRCFPWTNI 24 6 WTNITPPAL 11

TABLE XXX-V5 HLA-B2705-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 11; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 4 AILLLVLIF 17 5 ILLLVLIFL 17 6LLLVLIFLR 16 2 LEAILLLVL 14 8 LVLIFLRQR 14 3 EAILLLVLI 12 9 VLIFLRQRI 11

TABLE XXX-V6 HLA-B2705-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 9 PRSVFNLQI 19 5 KGLIPRSVF 17 2YSSKGLIPR 16 7 LIPRSVFNL 14 3 SSKGLIPRS 9

TABLE XXX-V7 HLA-B2705-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 15; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 8 AVGQMMSTM 13 4 WILVAVGQM 12 5ILVAVGQMM 11 3 YWILVAVGQ 6

TABLE XXX-V8 HLA-B2705-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 17; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 16 GHVFQTSIL 15 1 NYYWLPIMR 14 8MRNPITPTG 14 9 RNPITPTGH 14 11 PITPTGHVF 12 15 TGHVFQISI 11 19 FQTSILGAY10 2 YYWLPIMRN 8 4 WLPIMRNPI 7 7 IMRNPITPT 7 17 HVFQTSILG 7

TABLE XXX-V9 HLA-B2705-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 19; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 18 GYVLWASNI 15 13 QPATLGYVL 13 2WAMTALYPL 12 9 PLPTQPATL 12 11 PTQPATLGY 10 6 ALYPLPIQP 8 15 ATLGYVLWA 7

TABLE XXXI-V1 HLA-B2709-9merse-24P4C12 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123466789 score 332 QRIRIAIAL 23 179 GRCFPWTNV 22 250LRLVAGPLV 21 214 ARDISVKIF 20 436 QRSVFNLQI 20 144 NRNFCLPGV 19 330LRQRIRIAI 19 582 MRNIVRVVV 19 586 VRVVVLDKV 19 255 GPLVLVLIL 17 583RNIVHVVVL 17 251 RLVAGPLVL 16 683 RPYYMSKSL 16 78 GENKDKPYL 15 170FLLPSAPAL 15 334 IRIAIALLK 15 446 GVLGLFWTL 15 620 GRIPGLGKD 15 647GAYVIASGF 15 660 GMCVDTLFL 15 49 IVVGIVAWL 14 228 SWYWILVAL 14 234VALGVALVL 14 244 LLFILLLRL 14 317 AVLEAILLL 14 333 RIRIAIALL 14 452WTLNWVLAL 14 602 GKLLVVGGV 14 626 GKDFKSPHL 14 679 GSLDRPYYM 14 23FRGPIKNRS 13 34 DVICCVLFL 13 83 KPYLLYFNI 13 107 GLQCPTPQV 13 204GISGLIDSL 13 232 ILVALGVAL 13 236 LGVALVLSL 13 237 GVALVLSLL 13 240LVLSLLFIL 13 242 LSLLFILLL 13 253 VAGPLVLVL 13 291 SQLGFTTNL 13 311AALIVLAVL 13 322 ILLLMLIFL 13 357 YPLVTFVLL 13 358 PLVTFVLLL 13 369IAYWAMTAL 13 440 FNLQIYGVL 13 442 LQIYGVLGL 13 449 GLFWTLNWV 13 496RYHTGSLAF 13 500 GSLAFGALI 13 515 ARVILEYID 13 557 RNAYIMIAI 13 589VLVDKVTDL 13 15 KPVKYDPSF 12 38 CVLFLLFIL 12 45 ILGYIVVGI 12 56WLYGDPRQV 12 61 PRQVLYPRN 12 81 KDKPYLLYF 12 158 VITSLQQEL 12 164QELCPSFLL 12 258 VLVLILGVL 12 261 LILGVLGVL 12 287 GASISQLGE 12 308TWLAALIVL 12 316 LAVLEAILL 12 321 AILLLMLIF 12 328 IFLRQRIRI 12 355MFYPLVTFV 12 371 YWAMTALYL 12 414 LVNSSCPGL 12 432 KGLIQRSVF 12 434LIQRSVFNL 12 461 GQCVLAGAF 12 492 IRTLRYHTG 12 495 LRYHTGSLA 12 501SLAFGALIL 12 503 AFGALILTL 12 506 ALILTLVQI 12 518 ILEYIDHKL 12 553KFLNRNAYI 12 593 KVTDLLLFF 12 596 DLLLFFGKL 12 597 LLLFFGKLL 12 605LVVGGVGVL 12 608 GGVGVLSFF 12 621 RIPGLGKDF 12 637 YWLPIMTSI 12 666LFLCFLEDL 12 684 PYYMSKSLL 12 5 QRDEDDEAY 11 28 KNRSCTDVI 11 29NRSCTDVIC 11 32 CTDVICCVL 11 41 FLLFILGYI 11 42 LLFILGYIV 11 46LGYIVVGIV 11 67 PRNSTGAYC 11 79 ENKDKPYLL 11 87 LYFNIFSCI 11 100IISVAENGL 11 128 GKNEFSQTV 11 139 VFYTKNRNF 11 151 GVPWNMTVI 11 184WTNVTPPAL 11 217 ISVKIFEDF 11 225 FAQSWYWIL 11 230 YWILVALGV 11 238VALVLSLLF 11 239 ALVLSLLFI 11 249 LLHLVAGPL 11 257 LVLVLILGV 11 260VLILGVLGV 11 280 YRVLRDKGA 11 283 LRDKGASIS 11 285 DKGASISQL 11 297TNLSAYQSV 11 310 LAALIVLAV 11 314 IVLAVLEAI 11 319 LEAILLLML 11 351MMSTMFYPL 11 354 TMFYPLVTF 11 381 TSGQPQYVL 11 386 QYVLWASNI 11 427QGYSSKGLI 11 480 QDIPTFPLI 11 483 PTFPLISAF 11 509 LTLVQIARV 11 510TLVQIARVI 11 511 LVQIARVIL 11 526 LRGVQNPVA 11 534 ARCIMCCFK 11 537IMCCFKCCL 11 564 AIYGKNFCV 11 572 VSAKNAFML 11 591 LDKVTDLLL 11 592DKVTDLLLF 11 598 LLFFGKLLV 11 599 LFFGKLLVV 11 609 GVGVLSFFF 11 614SFFFFSGRI 11 617 FFSGRIPGL 11 631 SPHLNYYWL 11 634 LNYYWLPIM 11 643TSILGAYVI 11 653 SGFFSVFGM 11 658 VFGMCVDTL 11 663 VDTLFLCFL 11 675ERNNGSLDR 11 687 MSKSLLKIL 11

TABLE XXXI-V3 HLA-B2709-9mers-24P4C12 Each peptide is a portion of SEQID NO: 7; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 1 GRCFPWTNI 22 6 WINIIPPAL 11 9ITPPALPGI 11

TABLE XXXI-V5 B2709-9mers-24P4C12 Each peptide is a portion of SEQ IDNO: 11; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 4 AILLLVLIF 13 5 ILLLVLIFL 13 2LEAILLLVL 11 1 VLEAILLLV 10 3 EAILLLVLI 10 9 VLIFLRQRI 10

TABLE XXXI-V6 HLA-B2709-9mers-24P4C12 Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 9 PRSVFNLQI 20 5 KGLIPRSVF 12 7LIPRSVFNL 12 4 SKGLIPRSV 9

TABLE XXXI-V7 HLA-B2709-9mers-24P4C12 Each peptide is a portion of SEQID NO: 15; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 1 SWYWILVAV 12 4 WILVAVGQM 12 5ILVAVGQMM 10 8 AVGQMMSTM 9

TABLE XXXI-V8 HLA-B2709-9mers-24P4C12 Each peptide is a portion of SEQID NO: 17; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 16 GHVFQTSIL 14 8 MRNPITPTG 13 11PITPTGHVF 10 10 NPITPTGHV 9 4 WLPIMRNPI 8 15 TGHVFQTSI 8 20 QTSILGAYV 8

TABLE XXXI-V9 HLA-B2709-9mers-24P4C12 Each peptide is a portion of SEQID NO: 19; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 18 GYVLWASNI 14 2 WAMTALYPL 11 13QPATLGYVL 11 9 PLPTQPAIL 10 12 TQPATLGYV 8

TABLE XXXII-V1 HLA-B4402-9mers-24P4C12 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 164 QELCPSFLL 22 222 LEAILLLML 22 78GENKDKPYL 20 306 QETWLAALI 20 483 PTFPLISAF 20 317 AVLEAILLL 19 332QRIRIAIAL 19 503 AFGALILTL 18 506 ALILTLVQI 18 552 IKFLNRNAY 18 58YGDPRQVLY 17 170 FLLPSAPAL 17 214 ARDISVKIF 17 242 LSLLFILLL 17 583RNIVRVVVL 17 11 EAYGKPVKY 16 40 LFLLFILGY 16 48 YIVVGIVAW 16 81KDKPYLLYF 16 121 PEDPWTVGK 16 228 SWYWILVAL 16 253 VAGPLVLVL 16 254AGPLVLVLI 16 311 AALIVLAVL 16 320 EAILLLMLI 16 321 AILLLMLIF 16 363VLLLIQIAY 16 382 SGQPQYVLW 16 452 WTLNWVLAL 16 480 QDIPTEPLI 16 487LISAFIRTL 16 489 SAFIRTLRY 16 617 FFSGRIPGL 16 629 FKSPHLNYY 16 699NEAPPDNKK 16 34 DVICCVLFL 15 79 ENKDKPYLL 15 130 NEFSQIVGE 15 154WNMTVITSL 15 204 GISGLIDSL 15 234 VALGVALVL 15 241 VLSLLFILL 15 263LGVLGVLAY 15 278 EEYRVLRDK 15 294 GFTTNLSAY 15 354 TMFYPLVTF 15 370AYWAMTALY 15 399 CEKVPINTS 15 442 LQIYGVLGL 15 468 AFASFYWAF 15 477HKPQDIPTF 15 499 TGSLAFGAL 15 513 QIARVILEY 15 547 CLEKEIKEL 15 66YPRNSTGAY 14 80 NKDKPYLLY 14 84 PYLLYFNIF 14 93 SCILSSNII 14 104AENGLQCPT 14 193 PGITNDTTI 14 223 EDFAQSWYW 14 239 ALVLSLLFI 14 244LLFILLLRL 14 258 VLVLILGVL 14 261 LILGVLGVL 14 285 DKGASISQL 14 291SQLGFTTNL 14 301 AYQSVQETW 14 305 VQETWLAAL 14 308 TWLAALIVL 14 316LAVLEAILL 14 322 ILLLMLIFL 14 330 LRQRIRIAI 14 333 RIRIAIALL 14 356FYPLVTFVL 14 357 YPLVTFVLL 14 358 PLVTFVLLL 14 364 LLLICIAYW 14 418SCPGLMCVF 14 432 KGLIQRSVF 14 446 GVLGLFWTL 14 496 RYHTGSLAF 14 546WCLEKFIKF 14 558 NAYIMIAIY 14 573 SAKNAFMLL 14 577 AFMLLMRNI 14 592DKVTDLLLF 14 593 KVTDLLLFF 14 596 DLLLFFGKL 14 597 LLLFFGKLL 14 621RIPGLGKDF 14 841 IMTSILGAY 14 643 ISILGAYVI 14 651 IASGFFSVF 14 662CVDTLFLCF 14 671 LEDLERNNG 14 678 NGSLDRPYY 14 5 QRDEDDEAY 13 7DEDDEAYGK 13 32 CTDVICCVL 13 36 ICCVLFLLF 13 49 IVVGIVAWL 13 57LYGDPRQVL 13 77 MGENKDKPY 13 87 LYFNIFSCI 13 137 GEVFYTKNR 13 146NFCLPGVPW 13 174 SAPALGRCF 13 176 PALGRCFPW 13 184 WTNVTPPAL 13 187VTPPALPGI 13 200 TIQQGISGL 13 209 IDSLNARDI 13 213 NARDISVKI 13 232ILVALGVAL 13 237 GVALVLSLL 13 238 VALVLSLLF 13 251 RLVAGPLVL 13 255GPLVLVLIL 13 277 WEEYRVLRD 13 342 KEASKAVGQ 13 351 MMSTMFYPL 13 440FNLQIYGVL 13 443 QIYGVLGLF 13 448 LGLFWTLNW 13 461 GQCVLAGAF 13 466AGAFASFYW 13 501 SLAFGALIL 13 518 ILEYIDHKL 13 519 LEYIDHKLR 13 529VQNPVARQI 13 543 CCLWCLEKF 13 570 FCVSAKNAF 13 589 VVLDKVTDL 13 590VLDKVTDLL 13 605 LVVGGVGVL 13 631 SPHLNYYWL 13 637 YWLPIMTSI 13 648AYVIASGFF 13 674 LERNNGSLD 13 687 MSKSLLKIL 13 33 TDVICCVLF 12 35VICCVLFLL 12 38 CVLFLLFIL 12 50 VVGIVAWLY 12 100 IISVAENGL 12 132FSQTVGEVF 12 133 SQTVGEVFY 12 139 VFYTKNRNF 12 141 YTKNRNFCL 12 163QQELCPSFL 12 217 ISVKIFEDF 12 221 IFEDFAQSW 12 236 LGVALVLSL 12 240LVLSLLFIL 12 249 LLRLVAGPL 12 267 GVLAYGIYY 12 269 LAYGIYYCW 12 275YCWEEYRVL 12 287 GASISQLGF 12 314 IVLAVLEAI 12 326 MLIFLRQRI 12 328IFLRQRIRI 12 349 GQMMSTMFY 12 369 IAYWAMTAL 12 371 YWAMTALYL 12 406TSCNPTAHL 12 421 GLMCVFQGY 12 426 FQGYSSKGL 12 434 LIQRSVFNL 12 437RSVFNLQIY 12 450 LFWTLNWVL 12 457 VLALGQCVL 12 464 VLAGAFASF 12 479PQDIPTFPL 12 510 TLVQIARVI 12 511 LVQIARVIL 12 548 LEKFIKFLN 12 553KFLNRNAYI 12 557 RNAYIMIAI 12 562 MIAIYGKNF 12 572 VSAKNAFML 12 591LDKVTDLLL 12 607 VGGVGVLSF 12 608 GGVGVLSFF 12 610 VGVLSFFFF 12 630KSPHLNYYW 12 638 WLPIMTSIL 12 647 GAYVIASGF 12 658 VFGMCVDTL 12 659FGMCVDILF 12 660 GMCVDTLFL 12 663 VDTLFLCFL 12 666 LFLCFLEDL 12 673DLERNNGSL 12 677 NNGSLDRPY 12 683 RPYYMSKSL 12 686 YMSKSLLKI 12 10DEAYGKPVK 11 15 KPVKYDPSF 11 28 KNRSCTDVI 11 37 CCVLFLLFI 11 41FLLFILGYI 11 45 ILGYIVVGI 11 117 VSSCPEDPW 11 124 PWTVGKNEF 11 151GVPWNMTVI 11 197 NDTTIQQGI 11 201 IQQGISGLI 11 266 LGVLAYGIY 11 302YQSVQETWL 11 359 LVTFVLLLI 11 361 TFVLLLICI 11 379 LATSGQPQY 11 381TSGQPQYVL 11 436 QRSVFNLQI 11 444 IYGVLGLFW 11 465 LAGAFASFY 11 474WAFHKPQDI 11 484 TEPLISAFI 11 494 TLRYHTGSL 11 533 VARCIMCCF 11 538MCCFKCCLW 11 540 CFKCCLWCL 11 614 SFFFFSGRI 11 626 GKDFKSPHL 11 628DFKSPHLNY 11 684 PYYMSKSLL 11 19 YDPSFRGPI 10 83 KPYLLYFNI 10 88YFNIFSCIL 10 158 VITSLQQEL 10 162 LQQELCPSF 10 225 FAQSWYWIL 10 272GIYYCWEEY 10 315 VLAVLEAIL 10 348 VGQMMSTMF 10 386 QYVLWASNI 10 396SPGCEKVPI 10 414 LVNSSCPGL 10 500 GSLAFGALI 10 514 IARVILEYI 10 537IMCCFKCCL 10 544 CLWCLEKFI 10 555 LNRNAYIMI 10 609 GVGVLSFFF 10

TABLE XXXII-V3 HLA-B4402-9mers-24P4C12 Each peptide is a portion of SEQID NO: 7; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 6 WTNITPPAL 13 9 ITPPALPGI 13 1GRCFPWTNI 8 2 RCFPWTNIT 7 7 TNITPPALP 6 8 NITPPALPG 6

TABLE XXXII-V5 HLA-B4402-9mers-24P4C12 Each peptide is a portion of SEQID NO: 11 each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 2 LEAILLLVL 23 3 EAILLLVLI 17 4AILLLVLIF 17 5 ILLLVLIFL 14 9 VLIFLRQRI 12

TABLE XXXII-V6 HLA-B4402-9mers-24P4C12 Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 5 KGLIPRSVF 14 7 LIPRSVFNL 13 9PRSVFNLQI 11 6 GLIPRSVFN 8

TABLE XXXII-V7 HLA-84402-9mers-24P4C12 Each peptide is a portion of SEQID NO: 15; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 1 SWYWILVAV 6 3 YWILVAVGQ 6 8 AVGQMMSTM4 4 WILVAVGQM 3 2 WYWILVAVG 2

TABLE XXXII-V8 HLA-B4402-9mers-24P4C12 Each peptide is a portion of SEQID NO: 17; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 11 PITPTGHVF 15 19 FQTSILGAY 14 4WLPIMRNPI 11 16 GHVFQTSIL 11 15 TGHVFQTSI 8

TABLE XXXII-V9 HLA-B4402-9mers-24P4C12 Each peptide is a portion of SEQID NO: 19; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 11 PTQPATLGY 15 9 PLPTQPATL 4 2WAMTALYPL 13 14 PATLGYVLW 13 13 QPATLGYVL 12 18 GYVLWASNI 10 6 ALYPLPTQP8 15 ATLGYVLWA 7

TABLE XXXIII-V1 HLA-B5101-9mers-24P4C12 Each peptide is a portion of SEQID NO: 19; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 234 VALGVALVL 27 213 NARDISVKI 25 46LGYIVVGIV 24 83 KPYLLYFNI 24 311 AALIVLAVL 24 253 VAGPLVLVL 23 310LAALIVLAV 23 357 YPLVTFVLL 23 369 IAYWAMTAL 23 474 WAFHKPQDI 23 514IARVILEYI 23 683 RPYYMSKSL 22 254 AGPLVLVLI 21 255 GPLVLVLIL 21 320EAILLLVLI 21 396 SPGCEKVPI 21 427 QGYSSKGLI 21 11 EAYGKPVKY 20 193PGITNDTTI 20 316 LAVLEAILL 20 123 DPWTVGKNE 19 236 LGVALVLSL 18 314IVLAVLEAI 18 599 LFFGKLLVV 18 686 YMSKSLLKI 18 60 DPRQVLYPR 17 150PGVPWNMTV 17 225 FAQSWYWIL 17 261 LILGVLGVL 17 269 LAYGIYYCW 17 300SAYQSVQET 17 504 FGALILTLV 17 558 NAYIMIAIY 17 573 SAKNAFMLL 17 651IASGFFSVF 17 182 FPWTNVTPP 16 192 LPGITNDTT 16 328 IFLRQRIRI 16 355MFYPLVTFV 16 359 LVTFVLLLI 16 458 LALGQCVLA 16 502 LAFGALILT 16 505GALILTLVQ 16 510 TLVQIARVI 16 581 LMRNIVRVV 16 631 SPHLNYYWL 16 9DDEAYGKPV 15 45 ILGYIVVGI 15 56 WLYGDPRQV 15 110 CPTPQVCVS 15 120CPEDPWTVG 15 151 GVPWNMTVI 15 172 LPSAPALGR 15 224 DFAQSWYWI 15 275YCWEEYRVL 15 308 TWLAALIVL 15 336 IAIALLKEA 15 338 IALLKEASK 15 375TALYLATSG 15 485 EPLISAFIR 15 529 VQNPVARCI 15 564 AIYGKNFCV 15 582MRNIVRVVV 15 596 DLLLFFGKL 15 637 YWLPIMTSI 15 643 TSILGAYVI 15 647GAYVIASGF 15 700 EAPPDNKKR 15 20 DPSFRGPIK 14 41 ELLEILGYI 14 43LFILGYIVV 14 72 GAYCGMGEN 14 87 LYFNIFSCI 14 119 SCPEDPWTV 14 152VPWNMTVIT 14 188 TPPALPGIT 14 190 PALPGITND 14 209 IDSLNARDI 14 230YWILVALGV 14 238 VALVLSLLF 14 257 LVLVLILGV 14 409 NPTAHLVNS 14 411TAHLVNSSC 14 450 LFWTLNWVL 14 465 LAGAFASFY 14 467 GAFASFYWA 14 482IPTEPLISA 14 499 TGSLAFGAL 14 509 LTLVQIARV 14 576 NAFMLLMRN 14 586VRVVVLDKV 14 589 VVLDKVTDL 14 602 GKLLVVGGV 14 605 LVVGGVGVL 14 639LPIMTSILG 14 701 APPDNKKRK 14 702 PPDNKKRKK 14 19 YDPSFRGPI 13 28KNRSCTDVI 13 34 DVICCVLFL 13 54 VAWLYGDPR 13 66 YPRNSTGAY 13 112TPQVCVSSC 13 149 LPGVPWNMT 13 174 SAPALGRCF 13 176 PALGRCFPW 13 187VTPPALPGI 13 189 PPALPGITN 13 201 IQQGISGLI 13 239 ALVLSLLFI 13 252LVAGPLVLV 13 282 VLRDKGASI 13 285 DKGASISQL 13 293 LGFTTNLSA 13 322ILLLMLIFL 13 330 LRQRIRIAI 13 340 LLKEASKAV 13 343 EASKAVGQM 13 356FYPLVTFVL 13 361 TFVLLLICI 13 384 QPQYVLWAS 13 478 KPQDIPTFP 13 487LISAFIRTL 13 489 SAFIRTLRY 13 500 GSLAFGALI 13 506 ALILTLVQI 13 521YIDHKLRGV 13 531 NPVARCIMC 13 553 KFLNRNAYI 13 555 LNRNAYIMI 13 563AIIYGKNFC 13 578 FMLLMRNIV 13 580 LLMRNIVRV 13

TABLE XXXIII-V3 HLA-B5101-9mers-24P4C12 Each peptide is a portion of SEQID NO: 7; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 4 FPWTNITPP 15 9 ITPPALPGI 14 1GRCFPWTNI 11 6 WTNITPPAL 8

TABLE XXXIII-V5 HLA-B5101-9mers-24P4C12 Each peptide is a portion of SEQID NO: 11; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 3 EAILLLVLI 22 5 ILLLVLIFL 14 2LEAILLLVL 13 1 VLEAILLLV 12 9 VLIFLRQRI 12

TABLE XXXIII-V6 HLA-B5101-9mers-24P4C12 Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 8 IPRSVFNLQ 16 7 LIPRSVFNL 12 9PRSVFNLQI 12 5 KGLIPRSVF 11 4 SKGLIPRSV 10

TABLE XXXIII-V7 HLA-B5101-9mers-24P4C12 Each peptide is a portion of SEQID NO: 15; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 1 SWYWILVAV 14 7 VAVGQMMST 12 2WYWILVAVG 6 3 YWILVAVGQ 6

TABLE XXXIII-V8 HLA-B5101-9mers-24P4C12 Each peptide is a portion of SEQID NO: 17; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 10 NPITPTGHV 21 15 TGHVFQTSI 18 13TPTGHVFQT 14 4 WLPIMRNPI 13 5 LPIMRNPIT 13

TABLE XXXIII-V9 HLA-B5101-9mers-24P4C12 Each peptide is a portion of SEQID NO: 19; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score 13 QPATLGYVL 20 2 WAMTALYPL 18 5TALYPLPTQ 16 8 YPLPTQPAT 15 10 LPTQPATLG 14 12 TQPATLGYV 13 17 LGYVLWASN12 9 PLPTQPATL 11 14 PATLGYVLW 11 18 GYVLWASNI 11

TABLE XXXIV-V1 HLA-A1-10Omers-24P4C12 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 221 IFEDFAQSWY 25 488 ISAFIRTLRY 25 39VLFLLFILGY 23 58 YGDPRQVLYP 23 79 ENKDKPYLLY 23 262 ILGVLGVLAY 23 512VQIARVILEY 22 627 KDFKSPHLNY 21 132 FSQTVGEVFY 20 266 LGVLAYGIYY 20 362FVLLLICIAY 20 590 VLDKVTDLLL 20 594 VTDLLLFFGK 20 318 VLEAILLLML 19 32CTDVICCVLF 18 49 IVVGIVAWLY 18 378 YLATSGQPQY 18 420 PGLMCVFQGY 18 464VLAGAFASFY 18 10 DEAYGKPVKY 17 57 LYGDPRQVLY 17 121 PEDPWTVGKN 17 265VLGVLAYGIY 17 271 YGIYYCWEEY 17 276 CWEEYRVLRD 17 369 IAYWAMTALY 17 551FIKFLNRNAY 17 80 NKDKPYLLYF 18 348 VGQMMSTMFY 16 676 RNNGSLDRPY 16 677NNGSLDRPYY 16 4 KQRDEDDEAY 15 18 KYDPSFRGPI 15 65 LYPRNSTGAY 15 76GMGENKDKPY 15 214 ARDISVKIFE 15 293 LGFTTNLSAY 15 436 QRSVFNLQIY 15 479PQDIPTFPLI 15 557 RNAYIMIAIY 15 628 DFKSPHLNYY 15 640 PIMTSILGAY 15 664DTLFLCFLED 15 283 LRDKGASISQ 14 521 YIDHKLRGVQ 14 673 DLERNNGSLD 14 141YTKNRNFCLP 13 305 VQETWLAALI 13 382 SGQPQYVLWA 13 407 SCNPTAHLVN 13 518ILEYIDHKLR 13 547 GLEKFIKFLN 13 670 FLEDLERNNG 13 680 SLDRPYYMSK 13 7DEDDEAYGKP 12 35 VIGCVLFLLF 12 159 ITSLQQELCP 12 163 QQELCPSFLL 12 242LSLLFILLLR 12 618 FSGRIPGLGK 12 626 GKDFKSPHLN 12 698 KNEAPPDNKK 12

TABLE XXXIV-V3 HLA-A1-10mers-24P4C12 Each peptide is a portion of SEQ IDNO: 7; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 10 ITPPALPGIT 10 3 RCFPWTNITP 9 7WTNITPPALP 8 8 TNITPPALPG 6 9 NITPPALPGI 4

TABLE XXXIV-V5 HLA-A1-10mers-24P4C12 Each peptide is a portion of SEQ IDNO: 11; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 2 VLEAILLLVL 19 7 LLLVLIFLRQ 10 1AVLEAILLLV 9

TABLE XXXIV-V6 HLA-A1-10mers-24P4C12 Each peptide is a portion of SEQ IDNO: 13; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 10 PRSVFNLQIY 15 1 QGYSSKGLIP 7 4SSKGLIPRSV 7 9 IPRSVFNLQI 7

TABLE XXXIV-V7 HLA-A1-10mers-24P4C12 Each peptide is a portion of SEQ IDNO: 15; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 1 QSWYWILVAV 4 2 SWYWILVAVG 4 4YWILVAVGQM 3 5 WILVAVGQMM 2 6 ILVAVGQMMS 2 8 VAVGQMMSTM 2 9 AVGQMMSTMF 2

TABLE XXXIV-V8 HLA-A1-10mers-24P4C12 Each peptide is a portion of SEQ IDNO: 17; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 19 VFQTSILGAY 16 4 YWLPIMRNPI 7 13ITPTGHVFQT 7 21 QTSILGAYVI 7

TABLE XXXIV-V9 HLA-A1-10mers-24P4C12 Each peptide is a portion of SEQ IDNO: 19; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 11 LPTQPATLGY 21 12 PTQPATLGYV 10

TABLE XXXV-V1 HLA-A0201-10mers-24P4C12 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 235 ALGVALVLSL 29 44 FILGYIVVGI 28 232ILVALGVALV 28 243 SLLFILLLRL 28 309 WLAALIVLAV 28 579 MLLMRNIVRV 28 244LLFILLLRLV 27 260 VLILGVLGVL 27 433 GLIQRSVFNL 27 508 ILTLVQIARV 27 580LLMRNIVRVV 27 598 LLFFGKLLVV 27 48 YIVVGIVAWL 26 94 CILSSNIISV 26 239ALVLSLLFIL 26 241 VLSLLFILLL 26 251 RLVAGPLVLV 26 321 AILLLMLIFL 26 441NLQIYGVLGL 26 502 LAFGALILTL 26 517 VILEYIDHKL 26 603 KLLVVGGVGV 26 604LLVVGGVGVL 26 452 ILGYIVVGIV 25 25 LVAGPLVLVL 25 304 SVQETWLAAL 25 312ALIVLAVLEA 25 318 VLEAILLLML 25 486 PLISAFIRTL 25 657 SVFGMCVDTL 25 665TLFLCFLEDL 25 248 LLLRLVAGPL 24 259 LVLILGVLGV 24 310 LAALIVLAVL 24 339ALLKEASKAV 24 597 LLLFFGKLLV 24 41 FLLFILGYIV 23 42 LLFILGYIVV 23 56WLYGDPRQVL 23 231 WILVALGVAL 23 249 LLHLVAGPLV 23 256 PLVLVLILGV 23 313LIVLAVLEAI 23 315 VLAVLEAILL 23 438 SVFNLQIYGV 23 459 ALGQCVLAGA 23 686YMSKSLLKIL 23 99 NIISVAENGL 22 257 LVLVLILGVL 22 354 TMFYPLVTFV 22 413HLVNSSCPGL 22 449 GLFWTLNWVL 22 506 ALILTLVQIA 22 510 TLVQIARVIL 22 513QIARVILEYI 22 581 LMRNIVRVVV 22 585 IVRVVVLDKV 22 590 VLDKVTDLLL 22 199TTIQQGISGL 21 247 ILLLRLVAGP 21 253 VAGPLVLVLI 21 316 LAVLEAILLL 21 501SLAFGALILT 21 505 GALILTLVQI 21 641 IMTSILGAYV 21 86 LLYFNIFSCI 20 95ILSSNIISVA 20 191 ALPGITNDTT 20 238 VALVLSLLFI 20 261 LILGVLGVLA 20 314IVLAVLEAIL 20 325 LMLIFLRQRI 20 329 FLRQRIRIAI 20 350 QMMSTMFYPL 20 358PLVTFVLLLI 20 368 CIAYWAMTAL 20 393 NISSPGCEKV 20 554 FLNRNAYIMI 20 596DLLLFFGKLL 20 645 ILGAYVIASG 20 649 YVIASGFFSV 20 34 DVICCVLFLL 19 64VLYPRNSTGA 19 85 YLLYFNIFSC 19 186 NVTPPALPGI 19 233 LVALGVALVL 19 264GVLGVLAYGI 19 317 AVLEAILLLM 19 327 LIFLRQRIRI 19 335 RIAIALLKEA 19 351MMSIMFYPLV 19 357 YPLVTFVLLL 19 363 VLLLICIAYW 19 364 LLLICIAYWA 19 365LLICIAYWAM 19 380 ATSGQPQYVL 19 457 VLALGQCVLA 19 536 CIMCCFKCCL 19 588VVVLDKVTDL 19 633 HLNYYWLPIM 19 644 SILGAYVIAS 19 39 VLFLLFILGY 18 157TVITSLQQEL 18 203 QGISGLIDSL 18 208 LIDSLNARDI 18 240 LVLSLLFILL 18 246FILLLRLVAG 18 262 ILGVLGVLAY 18 281 RVLRDKGASI 18 322 ILLLMLIFLR 18 332QRIRIAIALL 18 360 VIFVLLLICI 18 388 VLWASNISSP 18 448 LGLFWTLNWV 18 493RTLRYHTGSL 18 525 KLRGVQNPVA 18 589 VVLDKVTDLL 18 616 FFFSGRIPGL 18 662CVDTLFLCFL 18 685 YYMSKSLLKI 18 130 NEFSQTVGEV 17 143 KNRNFCLPGV 17 148CLPGVPWNMT 17 170 FLLPSAPALG 17 211 SLNARDISVK 17 227 QSWYWILVAL 17 254AGPLVLVLIL 17 296 TTNLSAYQSV 17 324 LLMLIFLRQR 17 373 AMIALYLATS 17 481DIPTFPLISA 17 546 WCLEKFIKFL 17 563 IAIYGKNFCV 17 582 MRNIVRVVVL 17 40LFLLFILGYI 16 108 LQCPTPQVCV 16 118 SSCPEDPWTV 16 169 SFLLPSAPAL 16 200TIQQGISGLI 16 207 GLIDSLNARD 16 212 LNARDISVKI 16 236 LGVALVLSLL 16 292QLGFTTNLSA 16 307 ETWLAALIVL 16 319 LEAILLLMLI 16 337 AIALLKEASK 16 366LICIAYWAMT 16 405 NTSCNPTAHL 16 451 FWTLNWVLAL 16 456 WVLALGQCVL 16 458LALGQCVLAG 16 503 AFGALILTLV 16 509 LTLVQIARVI 16 637 YWLPIMTSIL 16 33TDVICCVLFL 15 36 ICCVLFLLFI 15 90 NIFSCILSSN 15 161 SLQQELCPSF 15 225FAQSWYWILV 15 234 VALGVALVLS 15 250 LRLVAGPLVL 15 284 RDKGASISQL 15 323LLLMLIFLRQ 15 340 LLKEASKAVG 15 378 YLATSGQPQY 15 379 LATSGQPQYV 15 430SSKGLIQRSV 15 464 VLAGAFASFY 15 498 HTGSLAFGAL 15 520 EYIDHKLRGV 15 539CCFKCCLWCL 15 601 FGKLLVVGGV 15 690 SLLKILGKKN 15 26 PIKNRSCTDV 14 30RSCTDVICCV 14 37 CCVLFLLFIL 14 102 SVAENGLQCP 14 149 LPGVPWNMTV 14 153PWNMTVITSL 14 162 LQQELCPSFL 14 165 ELCPSFLLPS 14 171 LLPSAPALGR 14 177ALGRCFPWIN 14 220 KIFEDFAQSW 14 273 IYYCWEEYRV 14 338 IALLKEASKA 14 353STMFYPLVTF 14 370 AYWAMTALYL 14 395 SSPGCEKVPI 14 416 NSSCPGLMCV 14 445YGVLGLFWTL 14 483 PTFPLISAFI 14 500 GSLAFGALIL 14 571 CVSAKNAFML 14 577AFMLLMRNIV 14 595 TDLLLFFGKL 14 606 VVGGVGVLSF 14 639 LPIMTSILGA 14 680SLDRPYYMSK 14 693 KILGKKNEAP 14 694 ILGKKNEAPP 14

TABLE XXXV-V3 HLA-A0201-10mers-24P4C12 Each peptide is a portion of SEQID NO: 7; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 9 NITPPALPGI 23 10 ITPPALPGIT 12

TABLE XXXV-V5 HLA-A0201-10mers-24P4C12 Each peptide is a portion of SEQID NO: 11; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 5 AILLLVLIFL 26 1 AVLEAILLLV 25 2VLEAILLLVL 25 3 LEAILLLVLI 18 6 ILLLVLIFLR 18 8 LLVLIFLRQR 16 9LVLIFLRQRI 16 7 LLLVLIFLRQ 15 10 VLIFLRQRIR 12

TABLE XXXV-V6 HLA-A0201-10mers-24P4C12 Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 7 GLIPRSVFNL 29 4 SSKGLIPRSV 15

TABLE XXXV-V7 HLA-A0201-10mers-24P4C12 Each peptide is a portion of SEQID NO: 15; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 1 QSWYWILVAV 4 2 SWYWILVAVG 4 4YWILVAVGQM 3 5 WILVAVGQMM 2 6 ILVAVGQMMS 2 8 VAVGQMMSTM 2 9 AVGQMMSTMF 2

TABLE XXXV-V8 HLA-A0201-10mers-24P4C12 Each peptide is a portion of SEQID NO: 17; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 4 YWLPIMRNPI 15 5 WLPIMRNPIT 15 18HVFQTSILGA 15 7 PIMRNPITPT 14 13 ITPTGHVFQT 14 8 IMRNPITPTG 13 21QTSILGAYVI 13 20 FQISILGAYV 12 15 PTGHVFQTSI 11 10 RNPITPTGHV 10 16TGHVFQTSIL 10 12 PITPTGHVFQ 8

TABLE XXXV-V9 HLA-A0201-10mers-24P4C12 Each peptide is a portion of SEQID NO: 19; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 9 YPLPTQPATL 20 2 YWAMTALYPL 19 7ALYPLPIQPA 19 12 PTQPATLGYV 17 16 ATLGYVLWAS 15 4 AMTALYPLPT 14 5MTALYPLPTQ 13 17 TLGYVLWASN 13 13 TQPATLGYVL 11 18 LGYVLWASNI 11 15PATLGYVLWA 9

TABLE XXXVI-V1 HLA-A0203-10mers-24P4C12 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 303 QSVQETWLAA 19 168 PSFLLPSAPA 18 330LRQRIRIAIA 18 459 ALGQCVLAGA 18 461 GQCVLAGAFA 18 304 SVQETWLAAL 17 3GKQRDEDDEA 10 46 LGYIVVGIVA 10 64 VLYPRNSTGA 10 95 ILSSNIISVA 10 166LCPSFLLPSA 10 182 FPWTNVTPPA 10 205 ISGLIDSLNA 10 217 ISVKIFEDFA 10 226AQSWYWILVA 10 230 YWILVALGVA 10 245 LFILLLRLVA 10 261 LILGVLGVLA 10 279EYRVLRDKGA 10 292 QLGFTTNLSA 10 302 YQSVQETWLA 10 308 TWLAALIVLA 10 312ALIVLAVLEA 10 328 IFLRQRIRIA 10 335 RIAIALLKEA 10 338 IALLKEASKA 10 361TFVLLLICIA 10 364 LLLICIAYWA 10 367 ICIAYWAMTA 10 371 YWAMTALYLA 10 382SGQPQYVLWA 10 403 PINTSCNPTA 10 450 LFWTLNWVLA 10 457 VLALGQCVLA 10 466AGAFASFYWA 10 481 DIPTEPLISA 10 494 TLRYHTGSLA 10 497 YHTGSLAFGA 10 506ALILTLVQIA 10 525 KLRGVQNPVA 10 550 KFIKFLNRNA 10 555 LNRNAYIMIA 10 565IYGKNFCVSA 10 568 KNFCVSAKNA 10 639 LPIMTSILGA 10 643 TSILGAYVIA 10 692LKILGKKNEA 10 4 KQRDEDDEAY 9 47 GYIVVGIVAW 9 65 LYPRNSTGAY 9 96LSSNIISVAE 9 167 CPSFLLPSAP 9 169 SFLLPSAPAL 9 183 PWTNVTPPAL 9 206SGLIDSLNAR 9 218 SVKIFEDFAQ 9 227 QSWYWILVAL 9 231 WILVALGVAL 9 246FILLLRLVAG 9 262 ILGVLGVLAY 9 280 YRVLRDKGAS 9 293 LGFTTNLSAY 9 309WLAALIVLAV 9 313 LIVLAVLEAI 9 329 FLRQRIRIAI 9 331 RQRIRIAIAL 9 336IAIALLKEAS 9 339 ALLKEASKAV 9 362 FVLLLICIAY 9 365 LLICIAYWAM 9 368CIAYWAMTAL 9 372 WAMIALYLAT 9 383 GQPQYVLWAS 9 404 INTSCNPTAH 9 451FWTLNWVLAL 9 458 LALGQCVLAG 9 460 LGQCVLAGAF 9 462 QCVLAGAFAS 9 467GAFASFYWAF 9 482 IPTFPLISAF 9 495 LRYHTGSLAF 9 498 HTGSLAFGAL 9 507LILTLVQIAR 9 526 LRGVQNPVAR 9 551 FIKFLNRNAY 9 556 NRNAYIMIAI 9 566YGKNFCVSAK 9 569 NFCVSAKNAF 9 640 PIMTSILGAY 9 644 SILGAYVIAS 9 693KILGKKNEAP 9

TABLE XXXVI-V3 HLA-A0203-10mers-24P4C12 Each peptide is a portion of SEQID NO: 7; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 5 FPWTNITPPA 10 6 PWTNITPPAL 9 7WTNITPPALP 8

TABLE XXXVI-V5 HLA-A0203-10mers-24P4C12 Pos 1234567890 scoreNoResultsFound.

TABLE XXXVI-V6 HLA-A0203-10mers-24P4C12 Pos 1234567890 scoreNoResultsFound.

TABLE XXXVI-V7 HLA-A0203-10mers-24P4C12 Each peptide is a portion of SEQID NO: 15; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 1 QSWYWILVAV 9 2 SWYWILVAVG 8

TABLE XXXVI-V8 HLA-A0203-10mers-24P4C12 Each peptide is a portion of SEQID NO: 17; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 18 HVFQTSILGA 10 19 VFQTSILGAY 9 20FQTSILGAYV 8

TABLE XXXVI-V9 HLA-A0203-10mers-24P4C12 Each peptide is a portion of SEQID NO: 19; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 7 ALYPLPTQPA 10 15 PATLGYVLWA 10 8LYPLPTQPAT 9 16 ATLGYVLWAS 9 9 YPLPTQPATL 8 17 TLGYVLWASN 8

TABLE XXXVII-V1 HLA-A3-10mers-24P4C12 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 333 RIRIAIALLK 32 211 SLNARDISVK 30 337AIALLKEASK 28 516 RVILEYIDHK 28 281 RVLRDKGASI 27 680 SLDRPYYMSK 27 464VLAGAFASFY 25 584 NIVRVVVLDK 24 621 RIPGLGKDFK 24 49 IVVGIVAWLY 23 463CVLAGAFASF 23 233 LVALGVALVL 22 262 ILGVLGVLAY 22 376 ALYLATSGQP 22 443QIYGVLGLFW 22 525 KLRGVQNPVA 22 587 RVVVLDKVTD 22 603 KLLVVGGVGV 22 56WLYGDPRQVL 21 63 QVLYPRNSTG 21 177 ALGRCFPWTN 21 564 AIYGKNFCVS 21 606VVGGVGVLSF 21 39 VLFLLFILGY 20 53 IVAWLYGDPR 20 171 LLPSAPALGR 20 251RLVAGPLVLV 20 252 LVAGPLVLVL 20 282 VLRDKGASIS 20 362 FVLLLICIAY 20 378YLATSGQPQY 20 544 CLWCLEKFIK 20 650 VIASGFFSVF 20 95 ILSSNIISVA 19 170FLLPSAPALG 19 191 ALPGITNDTT 19 237 GVALVLSLLF 19 248 LLLRLVAGPL 19 260VLILGVLGVL 19 261 LILGVLGVLA 19 298 NLSAYQSVQE 19 312 ALIVLAVLEA 19 314IVLAVLEAIL 19 317 AVLEAILLLM 19 322 ILLLMLIFLR 19 340 LLKEASKAVG 19 347AVGQMMSTMF 19 494 TLRYHTGSLA 19 605 LVVGGVGVLS 19 618 FSGRIPGLGK 19 645ILGAYVIASG 19 673 DLERNNGSLD 19 6 RDEDDEAYGK 18 64 VLYPRNSTGA 18 134QTVGEVFYTK 18 231 WILVALGVAL 18 235 ALGVALVLSL 18 247 ILLLRLVAGP 18 258VLVLILGVLG 18 324 LLMLIFLRQR 18 456 WVLALGQCVL 18 532 PVARCIMCCF 18 72GAYCGMGENK 17 86 LLYFNIFSCI 17 161 SLQQELCPSF 17 207 GLIDSLNARD 17 220KIFEDFAQSW 17 232 ILVALGVALV 17 249 LLRLVAGPLV 17 257 LVLVLILGVL 17 264GVLGVLAYGI 17 265 VLGVLAYGIY 17 292 QLGFTTNLSA 17 309 WLAALIVLAV 17 326MLIFLRQRIR 17 364 LLLICIAYWA 17 388 VLWASNISSP 17 392 SNISSPGCEK 17 486PLISAFIRTL 17 506 ALILTLVQIA 17 551 FIKFLNRNAY 17 580 LLMRNIVRVV 17 598LLFFGKLLVV 17 612 VLSFFFFSGR 17 624 GLGKDFKSPH 17 649 YVIASGFFSV 17 657SVFGMCVDTL 17 667 FLCFLEDLER 17 684 PYYMSKSLLK 17 689 KSLLKILGKK 17 9DDEAYGKPVK 16 44 FILGYIVVGI 16 126 TVGKNEFSQT 16 165 ELCPSFLLPS 16 243SLLFILLLRL 16 246 FILLLRLVAG 16 259 LVLILGVLGV 16 272 GIYYCWEEYR 16 304SVQETWLAAL 16 318 VLEAILLLML 16 339 ALLKEASKAV 16 363 VLLLICIAYW 16 453TLNWVLALGQ 16 457 VLALGQCVLA 16 459 ALGQCVLAGA 16 487 LISAFIRTLR 16 508ILTLVQIARV 16 518 ILEYIDHKLR 16 559 AYIMIAIYGK 16 566 YGKNFCVSAK 16 571CVSAKNAFML 16 579 MLLMRNIVRV 16 596 DLLLFFGKLL 16 640 PIMTSILGAY 16 690SLLKILGKKN 16 693 KILGKKNEAP 16 35 VICCVLFLLF 15 41 FLLFILGYIV 15 42LLFILGYIVV 15 107 GLQCPTPQVC 15 120 CPEDPWTVGK 15 180 RCFPWTNVTP 15 323LLLMLIFLRQ 15 329 FLRQRIRIAI 15 367 ICIAYWAMTA 15 369 IAYWAMTALY 15 423MCVFQGYSSK 15 446 GVLGLFWTLN 15 491 FIRTLRYHTG 15 507 LILTLVQIAR 15 510TLVQIARVIL 15 585 IVRVVVLDKV 15 597 LLLFFGKLLV 15 604 LLVVGGVGVL 15 688SKSLLKILGK 15 694 ILGKKNEAPP 15 697 KKNEAPPDNK 15 698 KNEAPPDNKK 15

TABLE XXXVII-V3 HLA-A3-10mers-24P4C12 Each peptide is a portion of SEQID NO: 7; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 3 RCFPWTNITP 11 9 NITPPALPGI 11 8TNITPPALPG 9 10 ITPPALPGIT 7 7 WTNITPPALP 5

TABLE XXXVII-V5 HLA-A3-10mers-24P4C12 Each peptide is a portion of SEQID NO: 11; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 1 AVLEAILLLV 19 2 VLEAILLLVL 19 6ILLLVLIFLR 19 8 LLVLIFLRQR 18 10 VLIFLRQRIR 17 7 LLLVLIFLRQ 15 5AILLLVLIFL 14 9 LVLIFLRQRI 14 4 EAILLLVLIF 11

TABLE XXXVII-V6 HLA-A3-10mers-24P4C12 Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 7 GLIPRSVFNL 16 5 SKGLIPRSVF 14 1QGYSSKGLIP 12 8 LIPRSVFNLQ 11 9 IPRSVFNLQI 11 6 KGLIPRSVFN 10 4SSKGLIPRSV 7

TABLE XXXVII-V7 HLA-A3-10mers-24P4C12 Each peptide is a portion of SEQID NO: 15; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 9 AVGQMMSTMF 19 6 ILVAVGQMMS 16 5WILVAVGQMM 14 7 LVAVGQMMST 14 2 SWYWILVAVG 12 8 VAVGQMMSTM 9

TABLE XXXVII-V8 HLA-A3-10mers-24P4C12 Each peptide is a portion of SEQID NO: 17; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 12 PITPTGHVFQ 15 11 NPITPTGHVF 14 18HVFQTSILGA 13 7 PIMRNPITPT 12 5 WLPIMRNPIT 11 1 LNYYWLPIMR 10 8IMRNPITPTG 10 21 QTSILGAYVI 10 9 MRNPITPTGH 9 6 LPIMRNPITP 8 19VFQTSILGAY 8

TABLE XXXVII-V9 HLA-A3-10mers-24P4C12 Each peptide is a portion of SEQID NO: 19; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 7 ALYPLPTQPA 20 17 TLGYVLWASN 15 10PLPTQPATLG 14 9 YPLPTQPATL 13 1 AYWAMTALYP 11 18 LGYVLWASNI 10 4AMTALYPLPT 9 11 LPTQPATLGY 9 13 TQPATLGYVL 9

TABLE XXXVIII-V1 HLA-A26-10mers-24P4C12 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 34 DVICCVLFLL 34 138 EVFYTKNRNF 32 307ETWLAALIVL 31 657 SVFGMCVDTL 28 199 TTIQQGISGL 26 304 SVQETWLAAL 26 588VVVLDKVTDL 26 592 DKVTDLLLFF 25 49 IVVGIVAWLY 24 606 VVGGVGVLSF 24 157TVITSLQQEL 23 252 LVAGPLVLVL 23 257 LVLVLILGVL 23 320 EAILLLMLIF 23 628DFKSPHLNYY 23 79 ENKDKPYLLY 22 353 STMFYPLVTF 22 362 FVLLLICIAY 22 662CVDTLFLCFL 22 672 EDLERNNGSL 22 48 YIVVGIVAWL 20 198 DTTIQQGISG 20 216DISVKIFEDF 20 240 LVLSLLFILL 20 293 LGFTTNLSAY 20 640 PIMTSILGAY 20 10DEAYGKPVKY 19 39 VLFLLFILGY 19 131 EFSQTVGEVF 19 233 LVALGVALVL 19 237GVALVLSLLF 19 347 AVGQMMSTMF 19 438 SVFNLQIYGV 19 463 CVLAGAFASF 19 498HTGSLAFGAL 19 512 VQIARVILEY 19 520 EYIDHKLRGV 19 571 CVSAKNAFML 19 589VVLDKVTDLL 19 33 TDVICCVLFL 18 203 QGISGLIDSL 18 314 IVLAVLEAIL 18 456WVLALGQCVL 18 481 DIPTFPLISA 18 486 PLISAFIRTL 18 493 RTLRYHTGSL 18 502LAFGALILTL 18 516 RVILEYIDHK 18 532 PVARCIMCCF 18 549 EKFIKFLNRN 18 609GVGVLSFFFF 18 99 NIISVAENGL 17 102 SVAENGLQCP 17 156 MTVITSLQQE 17 236LGVALVLSLL 17 260 VLILGVLGVL 17 316 LAVLEAILLL 17 317 AVLEAILLLM 17 321AILLLMLIFL 17 360 VTFVLLLICI 17 442 LQIYGVLGLF 17 596 DLLLFFGKLL 17 604LLVVGGVGVL 17 616 FFFSGRIPGL 17 664 DTLFLCFLED 17 665 TLFLCFLEDL 17 682DRPYYMSKSL 17 32 CTDVICCVLF 16 37 CCVLFLLFIL 16 123 DPWTVGKNEF 16 165ELCPSFLLPS 16 186 NVTPPALPGI 16 224 DFAQSWYWIL 16 239 ALVLSLLFIL 16 262ILGVLGVLAY 16 266 LGVLAYGIYY 16 332 QRIRIAIALL 16 359 LVTFVLLLIC 16 380ATSGQPQYVL 16 400 EKVPINTSCN 16 405 NTSCNPTAHL 16 424 CVFQGYSSKG 16 433GLIQRSVFNL 16 539 CCFKCCLWCL 16 593 KVTDLLLFFG 16

TABLE XXXVIII-V3 HLA-A26-10mers-24P4C12 Each peptide is a portion of SEQID NO: 7; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 6 PWTNITPPAL 10 9 NITPPALPGI 10 10ITPPALPGIT 10 7 WTNITPPALP 8 3 RCFPWTNITP 7 8 TNITPPALPG 6 4 CFPWTNITPP4

TABLE XXXVIII-V5 HLA-A26-10mers-24P4C12 Each peptide is a portion of SEQID NO: 11; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 4 EAILLLVLIF 27 1 AVLEAILLLV 17 5AILLLVLIFL 17 2 VLEAILLLVL 13

TABLE XXXVIII-V6 HLA-A26-10mers-24P4C12 Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 7 GLIPRSVFNL 17 10 PRSVFNLQIY 14 5SKGLIPRSVF 10

TABLE XXXVIII-V7 HLA-A26-10mers-24P4C12 Each peptide is a portion of SEQID NO: 15; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234561890 score 9 AVGQMMSTMF 19 7 LVAVGQMMST 11 4YWILVAVGQM 10

TABLE XXXVIII-V8 HLA-A26-10mers-24P4C12 Each peptide is a portion of SEQID NO: 17; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 18 HVFQTSILGA 19 19 VFQTSILGAY 16 11NPITPTGHVF 13 13 ITPTGHVFQT 13 16 TGHVFQTSIL 10 15 PTGHVFQTSI 9

TABLE XXXVIII-V9 HLA-A26-10mers-24P4C12 Each peptide is a portion of SEQID NO: 19; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 12 PTQPATLGYV 14 5 MTALYPLPTQ 13 16ATLGYVLWAS 13 2 YWAMTALYPL 12 11 LPTQPATLGY 12 9 YPLPTQPATL 10 13TQPATLGYVL 10 15 PATLGYVLWA 6

TABLE XXXIX-V1 HLA-B0702-10mers-24P4C12 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 357 YPLVTFVLLL 23 478 KPQDIPTFPL 23 683RPYYMSKSLL 21 182 FPWTNVTPPA 19 83 KPYLLYFNIF 18 192 LPGITNDTTI 18 482IPTFPLISAF 18 639 LPIMTSILGA 18 149 KPGVPWNMTV 17 252 LVAGPLVLVL 17 380ATSGQPQYVL 17 402 VPINTSCNPT 17 485 FPLISAFIRT 17 123 DPWTVGKNEF 16 235ALGVALVLSL 16 254 AGPLVLVLIL 15 370 AYWAMTALYL 15 659 FGMCVDTLFL 15 33TDVICCVLFL 14 56 WLYGDPRQVL 14 175 APALGRCFPW 14 233 LVALGVALVL 14 241VLSLLFILLL 14 331 RQRIRIAIAL 14 405 NTSCNPTAHL 14 451 FWTLNWVLAL 14 502LAFGALILTL 14 582 MRNIVRVVVL 14 590 VLDKVTDLLL 14 15 KPVKYDPSFR 13 60DPRQVLYPRN 13 66 YPRNSTGAYC 13 110 CPTPQVCVSS 13 120 CPEDPWTVGK 13 167CPSFLLPSAP 13 172 LPSAPALGRC 13 226 AQSWYWILVA 13 227 QSWYWILVAL 13 231WILVALGVAL 13 250 LRLVAGPLVL 13 284 RDKGASISQL 13 290 ISQLGFTTNL 13 301AYQSVQETWL 13 310 LAALIVLAVL 13 314 IVLAVLEAIL 13 318 VLEAILLLML 13 321AILLLMLIFL 13 350 QMMSTMFYPL 13 355 MFYPLVTFVL 13 356 FYPLVTFVLL 13 368CIAYWAMTAL 13 396 SPGCEKVPIN 13 441 NLQIYGVLGL 13 498 HTGSLAFGAL 13 500GSLAFGALIL 13 510 TLVQIARVIL 13 525 KLRGVQNPVA 13 571 CVSAKNAFML 13 572VSAKNAFMLL 13 657 SVFGMCVDTL 13 686 YMSKSLLKIL 13 20 DPSFRGPIKN 12 48YIVVGIVAWL 12 169 SFLLPSAPAL 12 183 PWTNVTPPAL 12 189 PPALPGITND 12 239ALVLSLLFIL 12 243 SLLFILLLRL 12 304 SVQEIWLAAL 12 307 ETWLAALIVL 12 309WLAALIVLAV 12 316 LAVLEAILLL 12 409 NFTAHLVNSS 12 419 CPGLMCVFQG 12 425VFQGYSSKGL 12 456 WVLALGQCVL 12 493 RTLRYHTGSL 12 581 LMRNIVRVVV 12 588VVVLDKVTDL 12 604 LLVVGGVGVL 12 606 VVGGVGVLSF 12 622 IPGLGKDFKS 12 637YWLPIMTSIL 12 662 CVDTLFLCFL 12 701 APPDNKKRKK 12 18 KYDPSFRGPI 11 25GPIKNRSCTD 11 31 SCTDVICCVL 11 44 FILGYIVVGI 11 77 MGENKDKPYL 11 78GENKDKPYLL 11 140 FYTKNRNFCL 11 152 VPWNMTVITS 11 153 PWNMTVITSL 11 162LQQELCPSFL 11 188 TPPALPGITN 11 224 DFAQSWYWIL 11 236 LGVALVLSLL 11 240LVLSLLFILL 11 248 LLLRLVAGPL 11 257 LVLVLILGVL 11 260 VLILGVLGVL 11 274YYCWEEYRVL 11 312 ALIVLAVLEA 11 315 VLAVLEAILL 11 332 QRIRIAIALL 11 384QPQYVLWASN 11 395 SSPGCEKVPI 11 413 HLVNSSCPGL 11 433 GLIQRSVFNL 11 435IQRSVFNLQI 11 439 VFNLQIYGVL 11 445 YGVLGLFWTL 11 449 GLFWTLNWVL 11 503AFGALILTLV 11 531 NPVARCIMCC 11 536 CIMCCFKCCL 11 539 CCFKCCLWCL 11 546WCLEKFIKFL 11 565 IYGKNFCVSA 11 589 VVLDKVTDLL 11 595 IDLLLFFGKL 11 616FFFSGRIPGL 11 625 LGKDFKSPHL 11 630 KSPHLNYYWL 11 672 EDLERNNGSL 11

TABLE XXXIX-V3 HLA-B0702-10mers-24P4C12 Each peptide is a portion of SEQID NO: 7; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 5 FPWTNITPPA 19 6 PWTNITPPAL 12 1LGRCFPWTNI 9

TABLE XXXIX-V5 HLA-B0702-10mers-24P4C12 Each peptide is a portion of SEQID NO: 11; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 2 VLEAILLLVL 14 5 AILLLVLIFL 13 1AVLEAILLLV 10 4 EAILLLVLIF 10 3 LEAILLLVLI 9 9 LVLIFLRQRI 7

TABLE XXXIX-V6 HLA-B0702-10mers-24P4C12 Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 9 IPRSVFNLQI 21 7 GLIPRSVFNL 12

TABLE XXXIX-V7 HLA-B0702-10mers-24P4C12 Each peptide is a portion of SEQID NO: 15; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 9 AVGQMMSTMF 10 1 QSWYWILVAV 9 8VAVGQMMSTM 8 4 YWILVAVGQM 7 7 LVAVGQMMST 7 5 WILVAVGQMM 6

TABLE XXXIX-V8 HLA-B0702-10mers-24P4C12 Each peptide is a portion of SEQID NO: 17; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 11 NPITPTGHVF 17 14 TPTGHVFQTS 13 16TGHVFQTSIL 11 6 LPIMRNPITP 10 4 YWLPIMRNPI 9 7 PIMRNPITPT 9 21QTSILGAYVI 9 10 RNPITPTGHV 8 13 ITPTGHVFQT 8 15 PTGHVFQTSI 8 18HVFQTSILGA 8

TABLE XXXIX-V9 HLA-B0702-10mers-24P4C12 Each peptide is a portion of SEQID NO: 19; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 9 YPLPTQPATL 22 11 LPTQPATLGY 13 14QPATLGYVLW 13 2 YWAMTALYPL 12 4 AMTALYPLPT 12 13 TQPATLGYVL 12 7ALYPLPTQPA 11

TABLE XL-V1 HLA-B08-10mers-24P4C12 Pos 1234567890 score NoResultsFound.

TABLE XL-V3 HLA-B08-10mers-24P4C12 Pos 1234567890 score NoResultsFound.

TABLE XL-V5 HLA-B08-10mers-24P4C12 Pos 1234567890 score NoResultsFound.

TABLE XL-V6 HLA-B08-10mers-24P4C12 Pos 1234567890 score NoResultsFound.

TABLE XL-V7 HLA-B08-10mers-24P4C12 Pos 1234567890 score NoResultsFound.

TABLE XL-V8 HLA-B08-10mers-24P4C12 Pos 1234567890 score NoResultsFound.

TABLE XL-V9 HLA-B08-10mers-24P4C12 Pos 1234567890 score NoResultsFound.

TABLE XLI-V1 HLA-B1510-10mers-24P4C12 Pos 1234567890 scoreNoResultsFound.

TABLE XLI-V3 HLA-B1510-10mers-24P4C12 Pos 1234567890 scoreNoResultsFound.

TABLE XLI-V5 HLA-B1510-10mers-24P4C12 Pos 1234567890 scoreNoResultsFound.

TABLE XLI-V6 HLA-B1510-10mers-24P4C12 Pos 1234567890 scoreNoResultsFound.

TABLE XLI-V7 HLA-B1510-10mers-24P4C12 Pos 1234567890 scoreNoResultsFound.

TABLE XLI-V8 HLA-B1510-10mers-24P4C12 Pos 1234567890 scoreNoResultsFound.

TABLE XLI-V9 HLA-B1510-10mers-24P4C12 Pos 1234567890 scoreNoResultsFound.

TABLE XLII-V1 HLA-B2705-10mers-24P4C12 Pos 1234567890 scoreNoResultsFound.

TABLE XLII-V3 HLA-B2705-10mers-24P4C12 Pos 1234567890 scoreNoResultsFound.

TABLE XLII-V5 HLA-B2705-10mers-24P4C12 Pos 1234567890 scoreNoResultsFound.

TABLE XLII-V6 HLA-B2705-10mers-24P4C12 Pos 1234567890 scoreNoResultsFound.

TABLE XLII-V7 HLA-B2705-10mers-24P4C12 Pos 1234567890 scoreNoResultsFound.

TABLE XLII-V8 HLA-B2705-10mers-24P4C12 Pos 1234567890 scoreNoResultsFound.

TABLE XLII-V9 HLA-B2705-10mers-24P4C12 Pos 1234567890 scoreNoResultsFound.

TABLE XLIII-V1 HLA-B2709-10mers-24P4C12 Pos 1234567890 scoreNoResultsFound.

TABLE XLIII V3-HLA-B2709-10mers- 24P4C12 Pos 1234567890 score No ResultsFound.

TABLE XLIII V5-HLA-B2709-10mers- 24P4C12 Pos 1234567890 score No ResultsFound.

TABLE XLIII V6-HLA-B2709-10mers- 24P4C12 Pos 1234567890 score No ResultsFound.

TABLE XLIII V7-HLA-B2709-10mers- 24P4C12 Pos 1234567890 score No ResultsFound.

TABLE XLIII V8-HLA-B2709-10mers- 24P4C12 Pos 1234567890 score No ResultsFound.

TABLE XLIII V9-HLA-B2709-10mers- 24P4C12 Pos 1234567890 score No ResultsFound.

TABLE XLIV V1-HLA-B4402- 10mers-24P4C12 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 10 DEAYGKPVKY 23 78 GENKDKPYLL 22 222FEDFAQSWYW 21 319 LEAILLLMLI 20 47 GYIVVGIVAW 19 332 QRIRIAIALL 18 486PLISAFIRTL 18 502 LAFGALILTL 18 620 GRIPGLGKDF 18 39 VLFLLFILGY 17 241VLSLLFILLL 17 254 AGPLVLVLIL 17 320 EAILLLMLIF 17 321 AILLLMLIFL 17 476FHKPQDIPTF 17 512 VQIARVILEY 17 699 NEAPPDNKKR 17 121 PEDPWTVGKN 16 169SFLLPSAPAL 16 199 TTIQQGISGL 16 203 QGISGLIDSL 16 260 VLILGVLGVL 16 293LGFTTNLSAY 16 307 ETWLAALIVL 16 316 LAVLEAILLL 16 380 ATSGQPQYVL 16 546WCLEKFIKFL 16 657 SVFGMCVDTL 16 34 DVICCVLFLL 15 65 LYPRNSTGAY 15 79ENKDKPYLLY 15 99 NIISVAENGL 15 104 AENGLQCPTP 15 138 EVFYTKNRNF 15 213NARDISVKIF 15 235 ALGVALVLSL 15 239 ALVLSLLFIL 15 278 EEYRVLRDKG 15 284RDKGASISQL 15 353 STMFYPLVTF 15 355 MFYPLVTFVL 15 356 FYPLVTFVLL 15 362FVLLLICIAY 15 363 VLLLICIAYW 15 370 AYWAMTALYL 15 417 SSCPGLMCVF 15 442LQIYGVLGLF 15 451 FWTLNWVLAL 15 482 IPTFPLISAF 15 561 IMIAIYGKNF 15 596DLLLFFGKLL 15 616 FFFSGRIPGL 15 637 YWLPIMTSIL 15 640 PIMTSILGAY 15 4KQRDEDDEAY 14 18 KYDPSFRGPI 14 80 NKDKPYLLYF 14 83 KPYLLYFNIF 14 130NEFSQTVGEV 14 131 EFSQTVGEVF 14 157 TVITSLQQEL 14 164 QELCPSFLLP 14 173PSAPALGRCF 14 175 APALGRCFPW 14 183 PWTNVTPPAL 14 220 KIFEDFAQSW 14 227QSWYWILVAL 14 231 WILVALGVAL 14 233 LVALGVALVL 14 240 LVLSLLFILL 14 243SLLFILLLRL 14 250 LRLVAGPLVL 14 252 LVAGPLVLVL 14 253 VAGPLVLVLI 14 262ILGVLGVLAY 14 304 SVQETWLAAL 14 331 RQRIRIAIAL 14 357 YPLVIFVLLL 14 431SKGLIQRSVF 14 433 GLIQRSVFNL 14 467 GAFASFYWAF 14 542 KCCLWCLEKF 14 545LWCLEKFIKF 14 551 FIKFLNRNAY 14 569 NFCVSAKNAF 14 589 VVLDKVTDLL 14 595TDLLLFFGKL 14 627 KDFKSPHLNY 14 629 FKSPHLNYYW 14 665 TLFLCFLEDL 14 686YMSKSLLKIL 14 7 DEDDEAYGKP 13 31 SCTDVICCVL 13 32 CTDVICCVLF 13 35VICCVLFLLF 13 49 IVVGIVAWLY 13 56 WLYGDPRQVL 13 57 LYGDPRQVLY 13 87LYFNIFSCIL 13 145 RNFCLPGVPW 13 153 PWNMTVITSL 13 186 NVTPPALPGI 13 237GVALVLSLLF 13 248 LLLRLVAGPL 13 257 LVLVLILGVL 13 271 YGIYYCWEEY 13 301AYQSVQETWL 13 310 LAALIVLAVL 13 315 VLAVLEAILL 13 327 LIFLRQRIRI 13 342KEASKAVGQM 13 347 AVGQMMSTMF 13 405 NTSCNPTAHL 13 425 VFQGYSSKGL 13 441NLQIYGVLGL 13 445 YGVLGLFWTL 13 447 VLGLFWTLNW 13 449 GLFWTLNWVL 13 460LGQCVLAGAF 13 478 KPQDIPTFPL 13 483 PTFPLISAFI 13 493 RTLRYHTGSL 13 495LRYHTGSLAF 13 498 HTGSLAFGAL 13 500 GSLAFGALIL 13 517 VILEYIDHKL 13 539CCFKCCLWCL 13 557 RNAYIMIAIY 13 582 MRNIVRVVVL 13 590 VLDKVTDLLL 13 591LDKVTDLLLF 13 592 DKVTDLLLFF 13 606 VVGGVGVLSF 13 659 FGMCVDTLFL 13 661MCVDTLFLCF 13 662 CVDTLFLCFL 13 671 LEDLERNNGS 13 672 EDLERNNGSL 13 682DRPYYMSKSL 13 33 TDVICCVLFL 12 37 CCVLFLLFIL 12 44 FILGYIVVGI 12 76GMGENKDKPY 12 123 DPWTVGKNEF 12 132 FSQTVGEVFY 12 150 PGVPWNMTVI 12 163QQELCPSFLL 12 216 DISVKIFEDF 12 223 EDFAQSWYWI 12 236 LGVALVLSLL 12 266LGVLAYGIYY 12 274 YYCWEEYRVL 12 277 WEEYRVLRDK 12 286 KGASISQLGF 12 290ISQLGFTTNL 12 300 SAYQSVQETW 12 306 QETWLAALIV 12 313 LIVLAVLEAI 12 318VLEAILLLML 12 329 FLRQRIRIAI 12 350 QMMSTMFYPL 12 358 PLVTFVLLLI 12 360VTFVLLLICI 12 368 CIAYWAMTAL 12 369 IAYWAMTALY 12 378 YLATSGQPQY 12 381TSGQPQYVLW 12 395 SSPGCEKVPI 12 420 PGLMCVFQGY 12 436 QRSVFNLQIY 12 439VFNLQIYGVL 12 443 QIYGVLGLFW 12 456 WVLALGQCVL 12 463 CVLAGAFASF 12 464VLAGAFASFY 12 488 ISAFIRTLRY 12 505 GALILTLVQI 12 509 LTLVQIARVI 12 510TLVQIARVIL 12 548 LEKFIKFLNR 12 556 NRNAYIMIAI 12 571 CVSAKNAFML 12 572VSAKNAFMLL 12 576 NAFMLLMRNI 12 588 VVVLDKVTDL 12 604 LLVVGGVGVL 12 628DFKSPHLNYY 12 630 KSPHLNYYWL 12 650 VIASGFFSVF 12 674 LERNNGSLDR 12 676RNNGSLDRPY 12 677 NNGSLDRPYY 12 685 YYMSKSLLKI 12 14 GKPVKYDPSF 11 27IKNRSCTDVI 11 40 LFLLFILGYI 11 48 YIVVGIVAWL 11 77 MGENKDKPYL 11 116CVSSCPEDPW 11 137 GEVFYTKNRN 11 161 SLQQELCPSF 11 162 LQQELCPSFL 11 208LIDSLNARDI 11 212 LNARDISVKI 11 221 IFEDFAQSWY 11 238 VALVLSLLFI 11 264GVLGVLAYGI 11 305 VQETWLAALI 11 314 IVLAVLEAIL 11 348 VGQMMSTMFY 11 413HLVNSSCPGL 11 479 PQDIPTFPLI 11 499 TGSLAFGALI 11 519 LEYIDHKLRG 11 528GVQNPVARCI 11 532 PVARCIMCCF 11 536 CIMCCFKCCL 11 537 IMCCFKCCLW 11 543CCLWCLEKFI 11 552 IKFLNRNAYI 11 607 VGGVGVLSFF 11 608 GGVGVLSFFF 11 609GVGVLSFFFF 11 625 LGKDFKSPHL 11 632 PHLNYYWLPI 11 642 MTSILGAYVI 11 646LGAYVIASGF 11 658 VFGMCVDTLF 11 683 RPYYMSKSLL 11

TABLE XLIV V3-HLA-B4402- 10mers-24P4C12 Each peptide is a portion of SEQID NO: 7; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 6 PWTNITPPAL 14 9 NITPPALPGI 13 1LGRCFPWTNI 8 3 RCFPWTNITP 7 8 TNITPPALPG 6

TABLE XLIV V5-HLA-B4402- 10mers-24P4C12 Each peptide is a portion of SEQID NO: 11; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 3 LEAILLLVLI 21 4 EAILLLVLIF 18 5AILLLVLIFL 17 2 VLEAILLLVL 13 9 LVLIFLRQRI 10

TABLE XLIV V6-HLA-B4402- 10mers-24P4C12 Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 7 GLIPRSVFNL 17 5 SKGLIPRSVF 14 10PRSVFNLQIY 12 9 IPRSVFNLQI 10

TABLE XLIV V7-HLA-B4402- 10mers-24P4C12 Each peptide is a portion of SEQID NO: 15; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 9 AVGQMMSTMF 13 4 YWILVAVGQM 6

TABLE XLIV V8-HLA-B4402- 10mers-24P4C12 Each peptide is a portion of SEQID NO: 17; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 11 NPITPTGHVF 17 4 YWLPIMRNPI 14 19VFQTSILGAY 14 16 TGHVFQTSIL 11 21 QTSILGAYVI 11 15 PTGHVFQTSI 8

TABLE XLIV V9-HLA-B4402- 10mers-24P4C12 Each peptide is a portion of SEQID NO: 19; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. Pos 1234567890 score 9 YPLPTQPATL 16 14 QPATLGYVLW 13 11LPTQPATLGY 12 13 TQPATLGYVL 12 2 YWAMTALYPL 11 18 LGYVLWASNI 9 16ATLGYVLWAS 8 7 ALYPLPTQPA 7

TABLE XLV V1-HLA-B5101-10mers- 24P4C12 Pos 1234567890 score No ResultsFound.

TABLE XLV V3-HLA-B5101-10mers- 24P4C12 Pos 1234567890 score No ResultsFound.

TABLE XLV V5-HLA-B5101- 10mers-24P4C12 Pos 1234567890 score No ResultsFound.

TABLE XLV V6-HLA-B5101- 10mers-24P4C12 Pos 1234567890 score No ResultsFound.

TABLE XLV V7-HLA-B5101- 10mers-24P4C12 Pos 1234567890 score No ResultsFound.

TABLE XLV V8-HLA-B5101- 10mers-24P4C12 Pos 1234567890 score No ResultsFound.

TABLE XLV V9-HLA-B5101- 10mers-24P4C12 Pos 1234567890 score No ResultsFound.

TABLE XLVI V1-HLA-DRB1-0101- 15mers-24P4C12 Each peptide is a portion ofSEQ ID NO: 3; each start position is specified, the length of peptide is15 amino acids, and the end position for each peptide is the startposition plus fourteen Pos 123456789012345 score 227 QSWYWILVALGVALV 39206 SGLIDSLNARDISVK 33 247 ILLLRLVAGPLVLVL 33 313 LIVLAVLEAILLLML 33 601FGKLLVVGGVGVLSF 33 246 FILLLRLVAGPLVLV 32 262 ILGVLGVLAYGIYYC 32 353STMFYPLVTFVLLLI 32 368 CIAYWAMTALYLATS 32 652 ASGFFSVFGMCVDTL 32 39VLFLLFILGYIVVGI 31 181 CFPWTNVTPPALPGI 31 277 WEEYRVLRDKGASIS 31 559AYIMIAIYGKNFCVS 31 639 LPIMTSILGAYVIAS 31 85 YLLYFNIFSCILSSN 30 89FNIFSCILSSNIISV 30 257 LVLVLILGVLGVLAY 30 259 LVLILGVLGVLAYGI 30 635NYYWLPIMTSILGAY 30 646 LGAYVIASGFFSVFG 30 235 ALGVALVLSLLFILL 29 345SKAVGQMMSTMFYPL 29 40 LFLLFILGYIVVGIV 28 242 LSLLFILLLRLVAGP 28 359LVTFVLLLICIAYWA 28 453 TLNWVLALGQCVLAG 28 612 VLSFFFFSGRIPGLG 28 640PIMTSILGAYVIASG 28 167 CPSFLLPSAPALGRC 27 243 SLLFILLLRLVAGPL 27 280YRVLRDKGASISQLG 27 362 FVLLLICIAYWAMTA 27 423 MCVFQGYSSKGLIQR 27 501SLAFGALILTLVQIA 27 575 KNAFMLLMRNIVRVV 27 129 KNEFSQTVGEVFYTK 26 230YWILVALGVALVLSL 26 254 AGPLVLVLILGVLGV 26 384 QPQYVLWASNISSPG 26 436QRSVFNLQIYGVLGL 26 437 RSVFNLQIYGVLGLF 26 448 LGLFWTLNWVLALGQ 26 492IRTLRYHTGSLAFGA 26 551 FIKFLNRNAYIMIAI 26 594 VTDLLLFFGKLLVVG 26 633HLNYYWLPIMTSILG 26 688 SKSLLKILGKKNEAP 26 44 FILGYIVVGIVAWLY 25 53IVAWLYGDPRQVLYP 25 62 RQVLYPRNSTGAYCG 25 90 NIFSCILSSNIISVA 25 228SWYWILVALGVALVL 25 231 WILVALGVALVLSLL 25 239 ALVLSLLFILLLRLV 25 293LGFTTNLSAYQSVQE 25 299 LSAYQSVQETWLAAL 25 304 SVQETWLAALIVLAV 25 319LEAILLLMLIFLRQR 25 326 MLIFLRQRIRIAIAL 25 337 AIALLKEASKAVGQM 25 354TMFYPLVTFVLLLIC 25 371 YWAMTALYLATSGQP 25 399 CEKVPINTSCNPTAH 25 451FWTLNWVLALGQCVL 25 454 LNWVLALGQCVLAGA 25 471 SFYWAFHKPQDIPTF 25 482IPTFPLISAFIRTLR 25 526 LRGVQNPVARCIMCC 25 583 RNIVRVVVLDKVTDL 25 603KLLVVGGVGVLSFFF 25 51 VGIVAWLYGDPRQVL 24 97 SSNIISVAENGLQCP 24 229WYWILVALGVALVLS 24 238 VALVLSLLFILLLRL 24 255 GPLVLVLILGVLGVL 24 256PLVLVLILGVLGVLA 24 279 EYRVLRDKGASISQL 24 307 ETWLAALIVLAVLEA 24 310LAALIVLAVLEAILL 24 383 GQPQYVLWASNISSP 24 420 PGLMCVFQGYSSKGL 24 459ALGQCVLAGAFASFY 24 506 ALILTLVQIARVILE 24 523 DHKLRGVQNPVARCI 24 569NFCVSAKNAFMLLMR 24 579 MLLMRNIVRVVVLDK 24 588 VVVLDKVTDLLLFFG 24 607VGGVGVLSFFFFSGR 24 644 SILGAYVIASGFFSV 24 660 GMCVDTLFLCFLEDL 24 47GYIVVGIVAWLYGDP 23 59 GDPRQVLYPRNSTGA 23 165 ELCPSFLLPSAPALG 23 166LCPSFLLPSAPALGR 23 241 VLSLLFILLLRLVAG 23 374 MTALYLATSGQPQYV 23 412AHLVNSSCPGLMCVF 23 507 LILTLVQIARVILEY 23 508 ILTLVQIARVILEYI 23 566YGKNFCVSAKNAFML 23 604 LLVVGGVGVLSFFFF 23 636 YYWLPIMTSILGAYV 23 33TDVICCVLFLLFILG 22 43 LFILGYIVVGIVAWL 22 86 LLYFNIFSCILSSNI 22 160TSLQQELCPSFLLPS 22 198 DTTIQQGISGLIDSL 22 312 ALIVLAVLEAILLLM 22 316LAVLEAILLLMLIFL 22 349 GQMMSTMFYPLVTFV 22 363 VLLLICIAYWAMTAL 22 419CPGLMCVFQGYSSKG 22 439 VFNLQIYGVLGLFWT 22 441 NLQIYGVLGLFWTLN 22 458LALGQCVLAGAFASF 22 481 DIPTFPLISAFIRTL 22 511 LVQIARVILEYIDHK 22 587RVVVLDKVTDLLLFF 22 598 LLFFGKLLVVGGVGV 22 655 FFSVFGMCVDTLFLC 22 689KSLLKILGKKNEAPP 22 138 EVFYTKNRNFCLPGV 21 151 GVPWNMTVITSLQQE 21 153PWNMTVITSLQQELC 21 203 QGISGLIDSLNARDI 21 300 SAYQSVQETWLAALI 21 329FLRQRIRIAIALLKE 21 331 RQRIRIAIALLKEAS 21 409 NPTAHLVNSSCPGLM 21 518ILEYIDHKLRGVQNP 21 548 LEKFIKFLNRNAYIM 21 606 VVGGVGVLSFFFFSG 21 10DEAYGKPVKYDPSFR 20 20 DPSFRGPIKNRSCTD 20 272 GIYYCWEEYRVLRDK 20 333RIRIAIALLKEASKA 20 449 GLFWTLNWVLALGQC 20 476 FHKPQDIPTFPLISA 20 543CCLWCLEKFIKFLNR 20 563 IAIYGKNFCVSAKNA 20 599 LFFGKLLVVGGVGVL 20 614SFFFFSGRIPGLGKD 20 634 LNYYWLPIMTSILGA 20 645 ILGAYVIASGFFSVF 20 656FSVFGMCVDTLFLCF 20 657 SVFGMCVDTLFLCFL 20 37 CCVLFLLFILGYIVV 19 38CVLFLLFILGYIVVG 19 82 DKPYLLYFNIFSCIL 19 122 EDPWTVGKNEFSQTV 19 179GRCFPWTNVTPPALP 19 184 WTNVTPPALPGITND 19 245 LFILLLRLVAGPLVL 19 271YGIYYCWEEYRVLRD 19 317 AVLEAILLLMLIFLR 19 323 LLLMLIFLRQRIRIA 19 336IAIALLKEASKAVGQ 19 369 IAYWAMTALYLATSG 19 411 TAHLVNSSCPGLMCV 19 442LQIYGVLGLFWTLNW 19 460 LGQCVLAGAFASFYW 19 495 LRYHTGSLAFGALIL 19 503AFGALILTLVQIARV 19 557 RNAYIMIAIYGKNFC 19 586 VRVVVLDKVTDLLLF 19 683RPYYMSKSLLKILGK 19 684 PYYMSKSLLKILGKK 19

TABLE XLVI V3-HLA-DRB1-0101- 15mers-24P4C12 Each peptide is a portion ofSEQ ID NO: 7; each start position is specified, the length of peptide is15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score 9 CFPWTNITPPALPGI 31 7GRCFPWTNITPPALP 19 12 WTNITPPALPGITND 19 10 FPWTNITPPALPGIT 18 14NITPPALPGITNDTT 16

TABLE XLVI V5-HLA-DRB1-0101- 15mers-24P4C12 Each peptide is a portion ofSEQ ID NO: 11; each start position is specified, the length of peptideis 15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score 2 LIVLAVLEAILLLVL 33 8LEAILLLVLIFLRQR 25 15 VLIFLRQRIRIAIAL 25 1 ALIVLAVLEAILLLV 22 5LAVLEAILLLVLIFL 22 6 AVLEAILLLVLIFLR 19 12 LLLVLIFLRQRIRIA 19 13LLVLIFLRQRIRIAI 18 7 VLEAILLLVLIFLRQ 17 11 ILLLVLIFLRQRIRI 17 14LVLIFLRQRIRIAIA 17 4 VLAVLEAILLLVLIF 16 10 AILLLVLIFLRQRIR 16

TABLE XLVI V6-HLA-DRB1-0101- 15mers-24P4C12 Each peptide is a portion ofSEQ ID NO: 13; each start position is specified, the length of peptideis 15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score 2 MCVFQGYSSKGLIPR 2715 PRSVFNLQIYGVLGL 26 7 GYSSKGLIPRSVFNL 24 4 VFQGYSSKGLIPRSV 16 10SKGLIPRSVFNLQIY 16 12 GLIPRSVFNLQIYGV 16 1 LMCVFQGYSSKGLIP 15 8YSSKGLIPRSVFNLQ 15

TABLE XLVI V7-HLA-DRB1-0101- 15mers-24P4C12 Each peptide is a portion ofSEQ ID NO: 15; each start position is specified, the length of peptideis 15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score 6 QSWYWILVAVGQMMS 3112 LVAVGQMMSTMFYPL 29 7 SWYWILVAVGQMMST 25 8 WYWILVAVGQMMSTM 24 9YWILVAVGQMMSTMF 24 1 FEDFAQSWYWILVAV 18 5 AQSWYWILVAVGQMM 16 11ILVAVGQMMSTMFYP 15

TABLE XLVI V8-HLA-DRB1-0101- 15mers-24P4C12 Each peptide is a portion ofSEQ ID NO: 17; each start position is specified, the length of peptideis 15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score 24 VFQTSILGAYVIASG 287 NYYWLPIMRNPITPT 24 23 HVFQTSILGAYVIAS 23 6 LNYYWLPIMRNPITP 20 5HLNYYWLPIMRNPIT 18 21 TGHVFQTSILGAYVI 18 3 SPHLNYYWLPIMRNP 17 8YYWLPIMRNPITPTG 17 13 IMRNPITPTGHVFQT 17 11 LPIMRNPITPTGHVF 16 12PIMRNPITPTGHVFQ 16 14 MRNPITPTGHVFQTS 16 26 QTSILGAYVIASGFF 16 9YWLPIMRNPITPTGH 15 18 ITPTGHVFQTSILGA 15 19 TPTGHVFQTSILGAY 14 20PTGHVFQTSILGAYV 14

TABLE XLVI V9-HLA-DRB1-0101- 15mers-24P4C12 Each peptide is a portion ofSEQ ID NO: 19; each start position is specified, the length of peptideis 15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score 4 CIAYWAMTALYPLPT 3210 MTALYPLPTQPATLG 30 22 TLGYVLWASNISSPG 26 21 ATLGYVLWASNISSP 24 7YWAMTALYPLPTQPA 23 13 LYPLPTQPATLGYVL 23 5 IAYWAMTALYPLPTQ 19 2LICIAYWAMTALYPL 17 1 LLICIAYWAMTALYP 16 16 LPTQPATLGYVLWAS 16 23LGYVLWASNISSPGC 16 24 GYVLWASNISSPGCE 16 9 AMTALYPLPTQPATL 15

TABLE XLVII V1-HLA-DRB1-0301- 15mers-24P4C12 Each peptide is a portionof SEQ ID NO: 3; each start position is specified, the length of peptideis 15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score 54 VAWLYGDPRQVLYPR 36586 VRVVVLDKVTDLLLF 31 667 FLCFLEDLERNNGSL 29 312 ALIVLAVLEAILLLM 28 97SSNIISVAENGLQCP 27 155 NMTVITSLQQELCPS 27 454 LNWVLALGQCVLAGA 27 549EKFIKFLNRNAYIMI 27 136 VGEVFYTKNRNFCLP 26 508 ILTLVQIARVILEYI 26 622IPGLGKDFKSPHLNY 26 376 ALYLATSGQPQYVLW 25 447 VLGLFWTLNWVLALG 25 279EYRVLRDKGASISQL 24 534 ARCIMCCFKCCLWCL 24 567 GKNFCVSAKNAFMLL 24 229WYWILVALGVALVLS 23 238 VALVLSLLFILLLRL 23 14 GKPVKYDPSFRGPIK 22 218SVKIFEDFAQSWYWI 22 219 VKIFEDFAQSWYWIL 22 235 ALGVALVLSLLFILL 22 241VLSLLFILLLRLVAG 22 360 VTFVLLLICIAYWAM 22 515 ARVILEYIDHKLRGV 22 594VTDLLLFFGKLLVVG 22 33 TDVICCVLFLLFILG 21 167 CPSFLLPSAPALGRC 21 192LPGITNDTTIQQGIS 21 237 GVALVLSLLFILLLR 21 239 ALVLSLLFILLLRLV 21 260VLILGVLGVLAYGIY 21 302 YQSVQETWLAALIVL 21 319 LEAILLLMLIFLRQR 21 431SKGLIQRSVFNLQIY 21 461 GQCVLAGAFASFYWA 21 587 RVVVLDKVTDLLLFF 21 590VLDKVTDLLLFFGKL 21 595 TDLLLFFGKLLVVGG 21 658 VFGMCVDTLFLCFLE 21 32CTDVICCVLFLLFIL 20 37 CCVLFLLFILGYIVV 20 46 LGYIVVGIVAWLYGD 20 47GYIVVGIVAWLYGDP 20 74 YCGMGENKDKPYLLY 20 76 GMGENKDKPYLLYFN 20 231WILVALGVALVLSLL 20 233 LVALGVALVLSLLFI 20 246 FILLLRLVAGPLVLV 20 250LRLVAGPLVLVLILG 20 255 GPLVLVLILGVLGVL 20 258 VLVLILGVLGVLAYG 20 313LIVLAVLEAILLLML 20 316 LAVLEAILLLMLIFL 20 323 LLLMLIFLRQRIRIA 20 338IALLKEASKAVGQMM 20 411 TAHLVNSSCPGLMCV 20 439 VFNLQIYGVLGLFWT 20 484TFPLISAFIRTLRYH 20 559 AYIMIAIYGKNFCVS 20 588 VVVLDKVTDLLLFFG 20 602GKLLVVGGVGVLSFF 20 604 LLVVGGVGVLSFFFF 20 691 LLKILGKKNEAPPDN 20 156MTVITSLQQELCPSF 19 159 ITSLQQELCPSFLLP 19 205 ISGLIDSLNARDISV 19 335RIAIALLKEASKAVG 19 348 VGQMMSTMFYPLVTF 19 366 LICIAYWAMTALYLA 19 385PQYVLWASNISSPGC 19 505 GALILILVQIARVIL 19 576 NAFMLLMRNIVRVVV 19 607VGGVGVLSFFFFSGR 19 626 GKDFKSPHLNYYWLP 19 638 WLPIMTSILGAYVIA 19 648AYVIASGFFSVFGMC 19 663 VDTLFLCFLEDLERN 19 668 LCFLEDLERNNGSLD 19 684PYYMSKSLLKILGKK 19 689 KSLLKILGKKNEAPP 19 3 GKQRDEDDEAYGKPV 18 61PRQVLYPRNSTGAYC 18 98 SNIISVAENGLQCPT 18 114 QVCVSSCPEDPWTVG 18 214ARDISVKIFEDFAQS 18 243 SLLFILLLRLVAGPL 18 263 LGVLGVLAYGIYYCW 18 327LIFLRQRIRIAIALL 18 345 SKAVGQMMSTMFYPL 18 462 QCVLAGAFASFYWAF 18 530QNPVARCIMCCFKCC 18 560 YIMIAIYGKNFCVSA 18 569 NFCVSAKNAFMLLMR 18 579MLLMRNIVRVVVLDK 18 585 IVRVVVLDKVTDLLL 18 655 FFSVFGMCVDTLFLC 18 656FSVFGMCVDTLFLCF 18 660 GMCVDTLFLCFLEDL 18 664 DTLFLCFLEDLERNN 18 284RDKGASISQLGFTTN 17 290 ISQLGFTTNLSAYQS 17 324 LLMLIFLRQRIRIAI 17 325LMLIFLRQRIRIAIA 17 353 STMFYPLVTFVLLLI 17 423 MCVFQGYSSKGLIQR 17 437RSVFNLQIYGVLGLF 17 485 FPLISAFIRTLRYHT 17 517 VILEYIDHKLRGVQN 17 519LEYIDHKLRGVQNPV 17 523 DHKLRGVQNPVARCI 17 542 KCCLWCLEKFIKFLN 17 545LWCLEKFIKFLNRNA 17 548 LEKFIKFLNRNAYIM 17 614 SFFFFSGRIPGLGKD 17 619SGRIPGLGKDFKSPH 17 670 FLEDLERNNGSLDRP 17 692 LKILGKKNEAPPDNK 17

TABLE XLVII V3-HLA-DRB1-0301- 15mers-24P4C12 Each peptide is a portionof SEQ ID NO: 7; each start position is specified, the length of peptideis 15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score 12 WTNITPPALPGITND 123 APALGRCFPWTNITP 10 9 CFPWTNITPPALPGI 10 7 GRCFPWTNITPPALP 8 6LGRCFPWTNITPPAL 7

TABLE XLVII V5-HLA-DRB1-0301- 15mers-24P4C12 Each peptide is a portionof SEQ ID NO: 11; each start position is specified, the length ofpeptide is 15 amino acids, and the end position for each peptide is thestart position plus fourteen. Pos 123456789012345 score 1ALIVLAVLEAILLLV 28 8 LEAILLLVLIFLRQR 21 2 LIVLAVLEAILLLVL 20 5LAVLEAILLLVLIFL 20 12 LLLVLIFLRQRIRIA 20 13 LLVLIFLRQRIRIAI 17 14LVLIFLRQRIRIAIA 17 4 VLAVLEAILLLVLIF 15 9 EAILLLVLIFLRQRI 15 10AILLLVLIFLRQRIR 13

TABLE XLVII V6-HLA-DRB1-0301- 15mers-24P4C12 Each peptide is a portionof SEQ ID NO: 13; each start position is specified, the length ofpeptide is 15 amino acids, and the end position for each peptide is thestart position plus fourteen. Pos 123456789012345 score 10SKGLIPRSVFNLQIY 22 2 MCVFQGYSSKGLIPR 17 8 YSSKGLIPRSVFNLQ 16 11KGLIPRSVFNLQIYG 12 1 LMCVFQGYSSKGLIP 11 15 PRSVFNLQIYGVLGL 10

TABLE XLVII V7-HLA-DRB1-0301- 15mers-24P4C12 Each peptide is a portionof SEQ ID NO: 15; each start position is specified, the length ofpeptide is 15 amino acids, and the end position for each peptide is thestart position plus fourteen. Pos 123456789012345 score 9YWILVAVGQMMSTMF 18 12 LVAVGQMMSTMFYPL 18 1 FEDFAQSWYWILVAV 16 8WYWILVAVGQMMSTM 13 10 WILVAVGQMMSTMFY 10 13 VAVGQMMSTMFYPLV 10

TABLE XLVII V8-HLA-DRB1-0301- 15mers-24P4C12 Each peptide is a portionof SEQ ID NO: 17; each start position is specified, the length ofpeptide is 15 amino acids, and the end position for each peptide is thestart position plus fourteen. Pos 123456789012345 score 22GHVFQTSILGAYVIA 17 8 YYWLPIMRNPITPTG 16 15 RNPITPTGHVFQTSI 14 26QTSILGAYVIASGFF 13 21 TGHVFQTSILGAYVI 12 10 WLPIMRNPITPTGHV 11 11LPIMRNPITPTGHVF 11 3 SPHLNYYWLPIMRNP 10 7 NYYWLPIMRNPITPT 10 14MRNPITPTGHVFQTS 9 19 TPTGHVFQTSILGAY 8

TABLE XLVII V9-HLA-DRB1-0301- 15mers-24P4C12 Each peptide is a portionof SEQ ID NO: 19; each start position is specified, the length ofpeptide is 15 amino acids, and the end position for each peptide is thestart position plus fourteen. Pos 123456789012345 score 2LICIAYWAMTALYPL 19 23 LGYVLWASNISSPGC 19 10 MTALYPLPTQPATLG 13 7YWAMTALYPLPTQPA 12 12 ALYPLPTQPATLGYV 12 13 LYPLPTQPATLGYVL 12 20PATLGYVLWASNISS 12 3 ICIAYWAMTALYPLP 10 14 YPLPTQPATLGYVLW 10 24GYVLWASNISSPGCE 10 5 IAYWAMTALYPLPTQ 9 16 LPTQPATLGYVLWAS 9

TABLE XLVIII V1-DR1-0401- 15mers-24P4C12 Each peptide is a portion ofSEQ ID NO: 3; each start position is specified, the length of peptide is15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score 85 YLLYFNIFSCILSSN 2889 FNIFSCILSSNIISV 28 243 SLLFILLLRLVAGPL 28 353 STMFYPLVTFVLLLI 28 469FASFYWAFHKPQDIP 28 548 LEKFIKFLNRNAYIM 28 575 KNAFMLLMRNIVRVV 28 635NYYWLPIMTSILGAY 28 54 VAWLYGDPRQVLYPR 26 98 SNIISVAENGLQCPT 26 153PWNMTVITSLQQELC 26 189 PPALPGITNDTTIQQ 26 192 LPGITNDTTIQQGIS 26 323LLLMLIFLRQRIRIA 26 337 AIALLKEASKAVGQM 26 385 PQYVLWASNISSPGC 26 419CPGLMCVFQGYSSKG 26 454 LNWVLALGQCVLAGA 26 508 ILTLVQIARVILEYI 26 523DHKLRGVQNPVARCI 26 579 MLLMRNIVRVVVLDK 26 16 PVKYDPSFRGPIKNR 22 38CVLFLLFILGYIVVG 22 82 DKPYLLYFNIFSCIL 22 86 LLYFNIFSCILSSNI 22 122EDPWTVGKNEFSQTV 22 138 EVFYTKNRNFCLPGV 22 181 CFPWTNVTPPALPGI 22 219VKIFEDFAQSWYWIL 22 227 QSWYWILVALGVALV 22 228 SWYWILVALGVALVL 22 272GIYYCWEEYRVLRDK 22 277 WEEYRVLRDKGASIS 22 292 QLGFTTNLSAYQSVQ 22 299LSAYQSVQETWLAAL 22 306 QETWLAALIVLAVLE 22 354 TMFYPLVTFVLLLIC 22 359LVTFVLLLICIAYWA 22 384 QPQYVLWASNISSPG 22 423 MCVFQGYSSKGLIQR 22 442LQIYGVLGLFWTLNW 22 448 LGLFWTLNWVLALGQ 22 453 TLNWVLALGQCVLAG 22 488ISAFIRTLRYHTGSL 22 501 SLAFGALILTLVQIA 22 557 RNAYIMIAIYGKNFC 22 633HLNYYWLPIMTSILG 22 646 LGAYVIASGFFSVFG 22 652 ASGFFSVFGMCVDTL 22 667FLCFLEDLERNNGSL 22 682 DRPYYMSKSLLKILG 22 14 GKPVKYDPSFRGPIK 20 39VLFLLFILGYIVVGI 20 40 LFLLFILGYIVVGIV 20 43 LFILGYIVVGIVAWL 20 97SSNIISVAENGLQCP 20 133 SQTVGEVFYTKNRNF 20 146 NFCLPGVPWNMTVIT 20 149LPGVPWNMTVITSLQ 20 155 NMTVITSLQQELCPS 20 156 MTVITSLQQELCPSF 20 198DTTIQQGISGLIDSL 20 202 QQGISGLIDSLNARD 20 206 SGLIDSLNARDISVK 20 216DISVKIFEDFAQSWY 20 229 WYWILVALGVALVLS 20 230 YWILVALGVALVLSL 20 233LVALGVALVLSLLFI 20 235 ALGVALVLSLLFILL 20 238 VALVLSLLFILLLRL 20 239ALVLSLLFILLLRLV 20 241 VLSLLFILLLRLVAG 20 242 LSLLFILLLRLVAGP 20 246FILLLRLVAGPLVLV 20 247 ILLLRLVAGPLVLVL 20 254 AGPLVLVLILGVLGV 20 255GPLVLVLILGVLGVL 20 257 LVLVLILGVLGVLAY 20 259 LVLILGVLGVLAYGI 20 262ILGVLGVLAYGIYYC 20 279 EYRVLRDKGASISQL 20 287 GASISQLGFTTNLSA 20 290ISQLGFTTNLSAYQS 20 307 ETWLAALIVLAVLEA 20 310 LAALIVLAVLEAILL 20 311AALIVLAVLEAILLL 20 312 ALIVLAVLEAILLLM 20 313 LIVLAVLEAILLLML 20 315VLAVLEAILLLMLIF 20 316 LAVLEAILLLMLIFL 20 319 LEAILLLMLIFLRQR 20 321AILLLMLIFLRQRIR 20 324 LLMLIFLRQRIRIAI 20 331 RQRIRIAIALLKEAS 20 333RIRIAIALLKEASKA 20 335 RIAIALLKEASKAVG 20 356 FYPLVTFVLLLICIA 20 363VLLLICIAYWAMTAL 20 364 LLLICIAYWAMTALY 20 371 YWAMTALYLATSGQP 20 374MTALYLATSGQPQYV 20 401 KVPINTSCNPTAHLV 20 420 PGLMCVFQGYSSKGL 20 436QRSVFNLQIYGVLGL 20 444 IYGVLGLFWTLNWVL 20 445 YGVLGLFWTLNWVLA 20 447VLGLFWTLNWVLALG 20 451 FWTLNWVLALGQCVL 20 479 PQDIPTFPLISAFIR 20 484TFPLISAFIRTLRYH 20 485 FPLISAFIRTLRYHT 20 505 GALILTLVQIARVIL 20 506ALILTLVQIARVILE 20 511 LVQIARVILEYIDHK 20 514 IARVILEYIDHKLRG 20 516RVILEYIDHKLRGVQ 20 542 KCCLWCLEKFIKFLN 20 545 LWCLEKFIKFLNRNA 20 549EKFIKFLNRNAYIMI 20 558 NAYIMIAIYGKNFCV 20 582 MRNIVRVVVLDKVTD 20 583RNIVRVVVLDKVTDL 20 586 VRVVVLDKVTDLLLF 20 588 VVVLDKVTDLLLFFG 20 594VTDLLLFFGKLLVVG 20 595 TDLLLFFGKLLVVGG 20 601 FGKLLVVGGVGVLSF 20 619SGRIPGLGKDFKSPH 20 639 LPIMTSILGAYVIAS 20 642 MTSILGAYVIASGFF 20 660GMCVDTLFLCFLEDL 20 668 LCFLEDLERNNGSLD 20 688 SKSLLKILGKKNEAP 20 90NIFSCILSSNIISVA 18 125 WTVGKNEFSQTVGEV 18 152 VPWNMTVITSLQQEL 18 166LCPSFLLPSAPALGR 18 195 ITNDTTIQQGISGLI 18 203 QGISGLIDSLNARDI 18 210DSLNARDISVKIFED 18 289 SISQLGFTTNLSAYQ 18 295 FTTNLSAYQSVQETW 18 342KEASKAVGQMMSTMF 18 373 AMTALYLATSGQPQY 18 398 GCEKVPINTSCNPTA 18 428GYSSKGLIQRSVFNL 18 433 GLIQRSVFNLQIYGV 18 476 FHKPQDIPTFPLISA 18 481DIPTFPLISAFIRTL 18 502 LAFGALILTLVQIAR 18 527 RGVQNPVARCIMCCF 18 568KNFCVSAKNAFMLLM 18 611 GVLSFFFFSGRIPGL 18 623 PGLGKDFKSPHLNYY 18 657SVFGMCVDTLFLCFL 18 669 CFLEDLERNNGSLDR 18 20 DPSFRGPIKNRSCTD 16 45ILGYIVVGIVAWLYG 16 53 IVAWLYGDPRQVLYP 16 55 AWLYGDPRQVLYPRN 16 63QVLYPRNSTGAYCGM 16 144 NRNFCLPGVPWNMTV 16 151 GVPWNMTVITSLQQE 16 167CPSFLLPSAPALGRC 16 222 FEDFAQSWYWILVAL 16 226 AQSWYWILVALGVAL 16 271YGIYYCWEEYRVLRD 16 326 MLIFLRQRIRIAIAL 16 368 CIAYWAMTALYLATS 16 369IAYWAMTALYLATSG 16 375 TALYLATSGQPQYVL 16 387 YVLWASNISSPGCEK 16 437RSVFNLQIYGVLGLF 16 449 GLFWTLNWVLALGQC 16 466 AGAFASFYWAFHKPQ 16 470ASFYWAFHKPQDIPT 16 471 SFYWAFHKPQDIPTF 16 473 YWAFHKPQDIPTFPL 16 482IPTFPLISAFIRTLR 16 518 ILEYIDHKLRGVQNP 16 543 CCLWCLEKFIKFLNR 16 563IAIYGKNFCVSAKNA 16 598 LLFFGKLLVVGGVGV 16 612 VLSFFFFSGRIPGLG 16 613LSFFFFSGRIPGLGK 16 614 SFFFFSGRIPGLGKD 16 634 LNYYWLPIMTSILGA 16 653SGFFSVFGMCVDTLF 16 664 DTLFLCFLEDLERNN 16 62 RQVLYPRNSTGAYCG 15 325LMLIFLRQRIRIAIA 15 327 LIFLRQRIRIAIALL 15 519 LEYIDHKLRGVQNPV 15 587RVVVLDKVTDLLLFF 15 32 CTDVICCVLFLLFIL 14 33 TDVICCVLFLLFILG 14 36ICCVLFLLFILGYIV 14 37 CCVLFLLFILGYIVV 14 42 LLFILGYIVVGIVAW 14 46LGYIVVGIVAWLYGD 14 47 GYIVVGIVAWLYGDP 14 48 YIVVGIVAWLYGDPR 14 51VGIVAWLYGDPRQVL 14 61 PRQVLYPRNSTGAYC 14 83 KPYLLYFNIFSCILS 14 84PYLLYFNIFSCILSS 14 88 YFNIFSCILSSNIIS 14 92 FSCILSSNIISVAEN 14 93SCILSSNIISVAENG 14 124 PWTVGKNEFSQTVGE 14 136 VGEVFYTKNRNFCLP 14 159ITSLQQELCPSFLLP 14 163 QQELCPSFLLPSAPA 14 169 SFLLPSAPALGRCFP 14 175APALGRCFPWTNVTP 14 184 WTNVTPPALPGITND 14 205 ISGLIDSLNARDISV 14 218SVKIFEDFAQSWYWI 14 231 WILVALGVALVLSLL 14 237 GVALVLSLLFILLLR 14 244LLFILLLRLVAGPLV 14 249 LLRLVAGPLVLVLIL 14 250 LRLVAGPLVLVLILG 14 256PLVLVLILGVLGVLA 14 258 VLVLILGVLGVLAYG 14 260 VLILGVLGVLAYGIY 14 263LGVLGVLAYGIYYCW 14 296 TTNLSAYQSVQETWL 14 302 YQSVQETWLAALIVL 14 322ILLLMLIFLRQRIRI 14 338 IALLKEASKAVGQMM 14 345 SKAVGQMMSTMFYPL 14 348VGQMMSTMFYPLVTF 14 349 GQMMSTMFYPLVTFV 14 352 MSTMFYPLVTFVLLL 14 357YPLVTFVLLLICIAY 14 360 VTFVLLLICIAYWAM 14 361 TFVLLLICIAYWAMT 14 362FVLLLICIAYWAMTA 14 366 LICIAYWAMTALYLA 14 376 ALYLATSGQPQYVLW 14 391ASNISSPGCEKVPIN 14 399 CEKVPINTSCNPTAH 14 411 TAHLVNSSCPGLMCV 14 412AHLVNSSCPGLMCVF 14 422 LMCVFQGYSSKGLIQ 14 432 KGLIQRSVFNLQIYG 14 439VFNLQIYGVLGLFWT 14 441 NLQIYGVLGLFWTLN 14 455 NWVLALGQCVLAGAF 14 457VLALGQCVLAGAFAS 14 462 QCVLAGAFASFYWAF 14 489 SAFIRTLRYHTGSLA 14 492IRTLRYHTGSLAFGA 14 499 IGSLAFGALILTLVQ 14 504 FGALILTLVQIARVI 14 509LTLVQIARVILEYID 14 515 ARVILEYIDHKLRGV 14 526 LRGVQNPVARCIMCC 14 534ARCIMCCFKCCLWCL 14 535 RCIMCCFKCCLWCLE 14 552 IKFLNRNAYIMIAIY 14 559AYIMIAIYGKNFCVS 14 576 NAFMLLMRNIVRVVV 14 578 FMLLMRNIVRVVVLD 14 585IVRVVVLDKVTDLLL 14 591 LDKVTDLLLFFGKLL 14 596 DLLLFFGKLLVVGGV 14 602GKLLVVGGVGVLSFF 14 603 KLLVVGGVGVLSFFF 14 604 LLVVGGVGVLSFFFF 14 607VGGVGVLSFFFFSGR 14 609 GVGVLSFFFFSGRIP 14 610 VGVLSFFFFSGRIPG 14 622IPGLGKDFKSPHLNY 14 631 SPHLNYYWLPIMTSI 14 636 YYWLPIMTSILGAYV 14 647GAYVIASGFFSVFGM 14 655 FFSVFGMCVDTLFLC 14 658 VFGMCVDTLFLCFLE 14 663VDTLFLCFLEDLERN 14 665 TLFLCFLEDLERNNG 14 678 NGSLDRPYYMSKSLL 14 684PYYMSKSLLKILGKK 14 689 KSLLKILGKKNEAPP 14

TABLE XLVIII V3-HLA-DR1-0401- 15mers-24P4C12 Each peptide is a portionof SEQ ID NO: 7; each start position is specified, the length of peptideis 15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score 9 CFPWTNITPPALPGI 22 3APALGRCFPWTNITP 14 12 WTNITPPALPGITND 14 4 PALGRCFPWTNITPP 12 5ALGRCFPWTNITPPA 12 8 RCFPWTNITPPALPG 12 13 TNITPPALPGITNDT 12 14NITPPALPGITNDTT 12 7 GRCFPWTNITPPALP 10

TABLE XLVIII V5-DR1-0401- 15mers-24P4C12 Each peptide is a portion ofSEQ ID NO: 11; each start position is specified, the length of peptideis 15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score 12 LLLVLIFLRQRIRIA 261 ALIVLAVLEAILLLV 20 2 LIVLAVLEAILLLVL 20 4 VLAVLEAILLLVLIF 20 5LAVLEAILLLVLIFL 20 8 LEAILLLVLIFLRQR 20 10 AILLLVLIFLRQRIR 20 13LLVLIFLRQRIRIAI 20 15 VLIFLRQRIRIAIAL 16 14 LVLIFLRQRIRIAIA 15 9EAILLLVLIFLRQRI 14 11 ILLLVLIFLRQRIRI 14 3 IVLAVLEAILLLVLI 12 6AVLEAILLLVLIFLR 12

TABLE XLVIII V6-HLA-DR1-0401- 15mers-24P4C12 Each peptide is a portionof SEQ ID NO: 13; each start position is specified, the length ofpeptide is 15 amino acids, and the end position for each peptide is thestart position plus fourteen. Pos 123456789012345 score 2MCVFQGYSSKGLIPR 22 15 PRSVFNLQIYGVLGL 20 12 GLIPRSVFNLQIYGV 18 1LMCVFQGYSSKGLIP 14 11 KGLIPRSVFNLQIYG 14 7 GYSSKGLIPRSVFNL 12 8YSSKGLIPRSVFNLQ 12 9 SSKGLIPRSVFNLQI 12

TABLE XLVIII V7-HLA-DR1-0401- 15mers-24P4C12 Each peptide is a portionof SEQ ID NO: 15; each start position is specified, the length ofpeptide is 15 amino acids, and the end position for each peptide is thestart position plus fourteen. Pos 123456789012345 score 9YWILVAVGQMMSTMF 26 6 QSWYWILVAVGQMMS 22 7 SWYWILVAVGQMMST 22 8WYWILVAVGQMMSTM 20 1 FEDFAQSWYWILVAV 16 5 AQSWYWILVAVGQMM 16 10WILVAVGQMMSTMFY 14 12 LVAVGQMMSTMFYPL 14

TABLE XLVIII V8-HLA-DR1-0401- 15mers-24P4C12 Each peptide is a portionof SEQ ID NO: 3; each start position is specified, the length of peptideis 17 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score 7 NYYWLPIMRNPITPT 28 5HLNYYWLPIMRNPIT 22 8 YYWLPIMRNPITPTG 20 15 RNPITPTGHVFQTSI 20 26QTSILGAYVIASGFF 20 18 ITPTGHVFQTSILGA 18 19 TPTGHVFQTSILGAY 18 3SPHLNYYWLPIMRNP 14 10 WLPIMRNPITPTGHV 14 11 LPIMRNPITPTGHVF 14 21TGHVFQTSILGAYVI 14

TABLE XLVIII V9-HLA-DR1-0401- 15mers-24P4C12 Each peptide is a portionof SEQ ID NO: 19; each start position is specified, the length ofpeptide is 15 amino acids, and the end position for each peptide is thestart position plus fourteen. Pos 123456789012345 score 10MTALYPLPTQPATLG 26 23 LGYVLWASNISSPGC 26 11 TALYPLPTQPATLGY 22 22TLGYVLWASNISSPG 22 7 YWAMTALYPLPTQPA 20 20 PATLGYVLWASNISS 20 5IAYWAMTALYPLPTQ 16 2 LICIAYWAMTALYPL 14 3 ICIAYWAMTALYPLP 12 15PLPTQPATLGYVLWA 12 21 ATLGYVLWASNISSP 12

TABLE XLIX V1-DRB1-1101- 15mers-24P4C12 Each peptide is a portion of SEQID NO: 3; each start position is specified, the length of peptide is 15amino acids, and the end position for each peptide is the start positionplus fourteen. Pos 123456789012345 score 243 SLLFILLLRLVAGPL 31 10DEAYGKPVKYDPSFR 26 20 DPSFRGPIKNRSCTD 26 668 LCFLEDLERNNGSLD 26 575KNAFMLLMRNIVRVV 25 613 LSFFFFSGRIPGLGK 25 226 AQSWYWILVALGVAL 23 228SWYWILVALGVALVL 23 277 WEEYRVLRDKGASIS 23 359 LVTFVLLLICIAYWA 23 448LGLFWTLNWVLALGQ 23 579 MLLMRNIVRVVVLDK 23 598 LLFFGKLLVVGGVGV 22 633HLNYYWLPIMTSILG 22 276 CWEEYRVLRDKGASI 21 338 IALLKEASKAVGQMM 21 508ILTLVQIARVILEYI 21 516 RVILEYIDHKLRGVQ 21 542 KCCLWCLEKFIKFLN 21 585IVRVVVLDKVTDLLL 21 685 YYMSKSLLKILGKKN 21 172 LPSAPALGRCFPWTN 20 334IRIAIALLKEASKAV 20 371 YWAMTALYLATSGQP 20 549 EKFIKFLNRNAYIMI 20 591LDKVTDLLLFFGKLL 20 619 SGRIPGLGKDFKSPH 20 689 KSLLKILGKKNEAPP 20 36ICCVLFLLFILGYIV 19 122 EDPWTVGKNEFSQTV 19 256 PLVLVLILGVLGVLA 19 259LVLILGVLGVLAYGI 19 310 LAALIVLAVLEAILL 19 353 STMFYPLVTFVLLLI 19 523DHKLRGVQNPVARCI 19 567 GKNFCVSAKNAFMLL 19 612 VLSFFFFSGRIPGLG 19 636YYWLPIMTSILGAYV 19 16 PVKYDPSFRGPIKNR 18 48 YIVVGIVAWLYGDPR 18 85YLLYFNIFSCILSSN 18 137 GEVFYTKNRNFCLPG 18 181 CFPWTNVTPPALPGI 18 227QSWYWILVALGVALV 18 244 LLFILLLRLVAGPLV 18 326 MLIFLRQRIRIAIAL 18 419CPGLMCVFQGYSSKG 18 469 FASFYWAFHKPQDIP 18 470 ASFYWAFHKPQDIRT 18 488ISAFIRTLRYHTGSL 18 489 SAFIRTLRYHTGSLA 18 597 LLLFFGKLLVVGGVG 18 41FLLFILGYIVVGIVA 17 45 ILGYIVVGIVAWLYG 17 71 TGAYCGMGENKDKPY 17 86LLYFNIFSCILSSNI 17 306 QETWLAALIVLAVLE 17 325 LMLIFLRQRIRIAIA 17 354TMFYPLVTFVLLLIC 17 369 IAYWAMTALYLATSG 17 384 QPQYVLWASNISSPG 17 442LQIYGVLGLFWTLNW 17 482 IPTFPLISAFIRTLR 17 501 SLAFGALILTLVQIA 17 548LEKFIKFLNRNAYIM 17 615 FFFFSGRIPGLGKDF 17 635 NYYWLPIMTSILGAY 17 652ASGFFSVFGMCVDTL 17 82 DKPYLLYFNIFSCIL 16 89 FNIFSCILSSNIISV 16 179GRCFPWTNVTPPALP 16 253 VAGPLVLVLILGVLG 16 299 LSAYQSVQETWLAAL 16 323LLLMLIFLRQRIRIA 16 368 CIAYWAMTALYLATS 16 387 YVLWASNISSPGCEK 16 490AFIRTLRYHTGSLAF 16 494 TLRYHTGSLAFGALI 16 506 ALILTLVQIARVILE 16 517VILEYIDHKLRGVQN 16 557 RNAYIMIAIYGKNFC 16 563 IAIYGKNFCVSAKNA 16 583RNIVRVVVLDKVTDL 16 646 LGAYVIASGFFSVFG 16 43 LFILGYIVVGIVAWL 15 44FILGYIVVGIVAWLY 15 47 GYIVVGIVAWLYGDP 15 54 VAWLYGDPRQVLYPR 15 73AYCGMGENKDKPYLL 15 153 PWNMTVITSLQQELC 15 156 MTVITSLQQELCPSF 15 195ITNDTTIQQGISGLI 15 207 GLIDSLNARDISVKI 15 242 LSLLFILLLRLVAGP 15 357YPLVTFVLLLICIAY 15 429 YSSKGLIQRSVFNLQ 15 485 FPLISAFIRTLRYHT 15 519LEYIDHKLRGVQNPV 15 527 RGVQNPVARCIMCCF 15 545 LWCLEKFIKFLNRNA 15 595TDLLLFFGKLLVVGG 15 600 FFGKLLVVGGVGVLS 15 603 KLLVVGGVGVLSFFF 15 681LDRPYYMSKSLLKIL 15

TABLE XLIX V3-HLA-DRB1-1101- 15mers-24P4C12 Each peptide is a portion ofSEQ ID NO: 7; each start position is specified, the length of peptide is15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score 9 CFPWTNITPPALPGI 18 7GRCFPWTNITPPALP 16 12 WTNITPPALPGITND 8

TABLE XLIX V5-HLA-DRB1-1101- 15mers-24P4C12 Each peptide is a portion ofSEQ ID NO: 11; each start position is specified, the length of peptideis 15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score 15 VLIFLRQRIRIAIAL 1814 LVLIFLRQRIRIAIA 17 12 LLLVLIFLRQRIRIA 16 10 AILLLVLIFLRQRIR 15 2LIVLAVLEAILLLVL 14 8 LEAILLLVLIFLRQR 14 13 LLVLIFLRQRIRIAI 14 1ALIVLAVLEAILLLV 13 5 LAVLEAILLLVLIFL 13 9 EAILLLVLIFLRQRI 13 11ILLLVLIFLRQRIRI 13

TABLE XLIX V6-HLA-DRB1-1101- 15mers-24P4C12 Each peptide is a portion ofSEQ ID NO: 13; each start position is specified, the length of peptideis 15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score 8 YSSKGLIPRSVFNLQ 15 1LMCVFQGYSSKGLIP 14 15 PRSVFNLQIYGVLGL 13 2 MCVFQGYSSKGLIPR 10 5FQGYSSKGLIPRSVF 10 3 CVFQGYSSKGLIPRS 9 11 KGLIPRSVFNLQIYG 9 6QGYSSKGLIPRSVFN 8 4 VFQGYSSKGLIPRSV 7 7 GYSSKGLIPRSVFNL 7

TABLE XLIX V7-HLA-DRB1-1101- 15mers-24P4C12 Each peptide is a portion ofSEQ ID NO: 15; each start position is specified, the length of peptideis 15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score 5 AQSWYWILVAVGQMM 23 6QSWYWILVAVGQMMS 18 9 YWILVAVGQMMSTMF 18 7 SWYWILVAVGQMMST 16 12LVAVGQMMSTMFYPL 12 1 FEDFAQSWYWILVAV 11

TABLE XLIX V8-HLA-DRB1-1101- 15mers-24P4C12 Each peptide is a portion ofSEQ ID NO: 17; each start position is specified, the length of peptideis 15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score 7 NYYWLPIMRNPITPT 24 5HLNYYWLPIMRNPIT 18 6 LNYYWLPIMRNPITP 17 15 RNPITPTGHVFQTSI 16 8YYWLPIMRNPITPTG 13 21 TGHVFQTSILGAYVI 13

TABLE XLIX V9-HLA-DRB1-1101- 15mers-24P4C12 Each peptide is a portion ofSEQ ID NO: 19; each start position is specified, the length of peptideis 15 amino acids, and the end position for each peptide is the startposition plus fourteen. Pos 123456789012345 score 4 CIAYWAMTALYPLPT 2210 MTALYPLPTQPATLG 18 22 TLGYVLWASNISSPG 17 7 YWAMTALYPLPTQPA 14 13LYPLPTQPATLGYVL 13 20 PATLGYVLWASNISS 12 23 LGYVLWASNISSPGC 12 24GYVLWASNISSPGCE 12 5 IAYWAMTALYPLPTQ 10 11 TALYPLPTQPATLGY 10

TABLE L Properties of 24P4C12 Bioinformatic Program URL Outcome ORF ORFfinder 6 to 2138 Protein length 710aa Transmembrane region TM Predhttp://www.ch.embnet.org/ 11TM, 39-59, 86-104, 231-250, 252-273, 309-330, 360-380, 457-474, 497-515, 559-581, 604- 626, 641-663 HMMTophttp://www.enzim.hu/hmmtop/ 11TM, 35-59 84-104 231- 250 257-277 308-330355-377 456-475 500-519 550-572 597-618 649-671 Sosuihttp://www.genome.ad.jp/SOSui/ 13TM, 34-65, 86-108, 145-167, 225-247,307- 329, 357-379, 414-436, 447-469, 501-523, 564- 586, 600-622, 644-666TMHMM http://www.cbs.dtu.dk/services/TMHMM 10TM, 36-58, 228-250,252-274, 308-330, 356- 378, 454-476, 497-519, 559-581, 597-619 SignalPeptide Signal P http://www.cbs.dtu.dk/services/SignalP/ no pI pI/MWtool http://www.expasy.ch/tools/ 8.9 pI Molecular weight pI/MW toolhttp://www.expasy.ch/tools/ 79.3 kD Localization PSORThttp://psort.nibb.ac.jp/ 80% Plasma Membrane, 40% Golgi PSORT IIhttp://psort.nibb.ac.jp/ 65% Plasma Membrane, 38% endoplasmic reticulumMotifs Pfam http://www.sanger.ac.uk/Pfam/ DUF580, uknown function Printshttp://www.biochem.ucl.ac.uk/ Blocks http://www.blocks.fhcrc.org/ Anionexchanger family 313-359 Prosite http://www.prosite.org/ CYS-RICH536-547

TABLE LI Exon compositions of 24P4C12 v.1 Exon number Start End Length 11 45 45 2 46 94 49 3 95 168 74 4 169 247 79 5 248 347 100 6 348 473 1267 474 534 61 8 535 622 88 9 623 706 84 10 707 942 236 11 943 1042 100 121043 1135 93 13 1136 1238 103 14 1239 1492 254 15 1493 1587 95 16 15881691 104 17 1692 1765 74 18 1766 1836 71 19 1837 1931 95 20 1932 2016 8521 2017 2573 557

TABLE LII Nucleotide sequence of transcript variant 24P4C12 v.7 (SEQ IDNO: 94) gagccatggg gggaaagcag cgggacgagg atgacgaggc ctacgggaagccagtcaaat 60 acgacccctc ctttcgaggc cccatcaaga acagaagctg cacagatgtcatctgctgcg 120 tcctcttcct gctcttcatt ctaggttaca tcgtggtggg gattgtggcctggttgtatg 180 gagacccccg gcaagtcctc taccccagga actctactgg ggcctactgtggcatggggg 240 agaacaaaga taagccgtat ctcctgtact tcaacatctt cagctgcatcctgtccagca 300 acatcatctc agttgctgag aacggcctac agtgccccac accccaggtgtgtgtgtcct 360 cctgcccgga ggacccatgg actgtgggaa aaaacgagtt ctcacagactgttggggaag 420 tcttctatac aaaaaacagg aacttttgtc tgccaggggt accctggaatatgacggtga 480 tcacaagcct gcaacaggaa ctctgcccca gtttcctcct cccctctgctccagctctgg 540 ggcgctgctt tccatggacc aacgttactc caccggcgct cccagggatcaccaatgaca 600 ccaccataca gcaggggatc agcggtctta ttgacagcct caatgcccgagacatcagtg 660 ttaagatctt tgaagatttt gcccagtcct ggtattggat tcttgtggctgtgggacaga 720 tgatgtctac catgttctac ccactggtca cctttgtcct cctcctcatctgcattgcct 780 actgggccat gactgctctg tacctggcta catcggggca accccagtatgtgctctggg 840 catccaacat cagctccccc ggctgtgaga aagtgccaat aaatacatcatgcaacccca 900 cggcccacct tgtgaactcc tcgtgcccag ggctgatgtg cgtcttccagggctactcat 960 ccaaaggcct aatccaacgt tctgtcttca atctgcaaat ctatggggtcctggggctct 1020 tctggaccct taactgggta ctggccctgg gccaatgcgt cctcgctggagcctttgcct 1080 ccttctactg ggccttccac aagccccagg acatccctac cttccccttaatctctgcct 1140 tcatccgcac actccgttac cacactgggt cattggcatt tggagccctcatcctgaccc 1200 ttgtgcagat agcccgggtc atcttggagt atattgacca caagctcagaggagtgcaga 1260 accctgtagc ccgctgcatc atgtgctgtt tcaagtgctg cctctggtgtctggaaaaat 1320 ttatcaagtt cctaaaccgc aatgcataca tcatgatcgc catctacgggaagaatttct 1380 gtgtctcagc caaaaatgcg ttcatgctac tcatgcgaaa cattgtcagggtggtcgtcc 1440 tggacaaagt cacagacctg ctgctgttct ttgggaagct gctggtggtcggaggcgtgg 1500 gggtcctgtc cttctttttt ttctccggtc gcatcccggg gctgggtaaagactttaaga 1560 gcccccacct caactattac tggctgccca tcatgacctc catcctgggggcctatgtca 1620 tcgccagcgg cttcttcagc gttttcggca tgtgtgtgga cacgctcttcctctgcttcc 1680 tggaagacct ggagcggaac aacggctccc tggaccggcc ctactacatgtccaagagcc 1740 ttctaaagat tctgggcaag aagaacgagg cgcccccgga caacaagaagaggaagaagt 1800 gacagctccg gccctgatcc aggactgcac cccaccccca ccgtccagccatccaacctc 1860 acttcgcctt acaggtctcc attttgtggt aaaaaaaggt tttaggccaggcgccgtggc 1920 tcacgcctgt aatccaacac tttgagaggc tgaggcgggc ggatcacctgagtcaggagt 1980 tcgagaccag cctggccaac atggtgaaac ctccgtctct attaaaaatacaaaaattag 2040 ccgagagtgg tggcatgcac ctgtcatccc agctactcgg gaggctgaggcaggagaatc 2100 gcttgaaccc gggaggcaga ggttgcagtg agccgagatc gcgccactgcactccaacct 2160 gggtgacaga ctctgtctcc aaaacaaaac aaacaaacaa aaagattttattaaagatat 2220 tttgttaact cagtaaaaaa aaaaaaaaaa a 2251

TABLE LIV Peptide sequences of protein coded by 24P4C12 v.7 (SEQ ID NO:97) MGGKQRDEDD EAYGKPVKYD PSFRGPIKNR SCTDVICCVL FLLFILGYIV VGIVAWLYGD 60PRQVLYPRNS TGAYCGMGEN KDKPYLLYFN IFSCILSSNI ISVAENGLQC PTPQVCVSSC 120PEDPWTVGKN EFSQTVGEVF YTKNRNFCLP GVPWNMTVIT SLQQELCPSF LLPSAPALGR 180CFPWTNVTPP ALPGITNDTT IQQGISGLID SLNARDISVK IFEDFAQSWY WILVAVGQMM 240STMFYPLVTF VLLLICIAYW AMTALYLATS GQPQYVLWAS NISSPGCEKV PINTSCNPTA 300HLVNSSCPGL MCVFQGYSSK GLIQRSVFNL QIYGVLGLFW TLNWVLALGQ CVLAGAFASF 360YWAFHKPQDI PTFPLISAFI RTLRYHTGSL AFGALILTLV QIARVILEYI DHKLRGVQNP 420VARCIMCCFK CCLWCLEKFI KFLNRNAYIM IAIYGKNFCV SAKNAFMLLM RNIVRVVVLD 480KVTDLLLFFG KLLVVGGVGV LSFFFFSGRI PGLGKDFKSP HLNYYWLPIM TSILGAYVIA 540SGFFSVFGMC VDTLFLCFLE DLERNNGSLD RPYYMSKSLL KILGKENEAP PDNKKRKK 598

TABLE LV Amino acid sequence alignment of 24P4C12v.1 v.1 (SEQ ID NO: 98)and 24P4C12 v.7 (SEQ ID NO: 99). Score = 1195 bits (3091), Expect= 0.0Identities = 598/710 (84%), Positives = 598/710 (84%), Gaps= 112/710 (15%) 24P4C12v.1: 1MGGKQRDEDDEAYGKPVKYDPSFRGPIKNRSCTDVICCVLFLLFILGYIVVGIVAWLYGD 60MGGKQRDEDDEAYGKPVKYDPSFRGPIKNRSCTDVICCVLFLLFILGYIVVGIVAWLYGD 24P4C12v.7:1 MGGKQRDEDDEAYGKPVKYDPSFRGPIKNRSCTDVICCVLFLLFILGYIVVGIVAWLYGD 6024P4C12v.1: 61PRQVLYPRNSTGAYCGMGENKDKPYLLYFNIFSCILSSNIISVAENGLQCPTPQVCVSSC 120PRQVLYPRNSTGAYCGMGENKDKPYLLYFNIFSCILSSNIISVAENGLQCPTPQVCVSSC 24P4C12v.7:61 PRQVLYPRNSTGAYCGMGENKDKPYLLYFNIFSCILSSNIISVAENGLQCPTPQVCVSSC 12024P4C12v.1: 121PEDPWTVGKNEFSQTVGEVFYTKNRNFCLPGVPWNMTVITSLQQELCPSFLLPSAPALGR 180PEDPWTVGKNEFSQTVGEVFYTKNRNFCLPGVPWNMTVITSLQQELCPSFLLPSAPALGR 24P4C12v.7:121 PEDPWTVGKNEFSQTVGEVFYTKNRNFCLPGVPWNMTVITSLQQELCPSFLLPSAPALGR 18024P4C12v.1: 181CFPWTNVTPPALPGITNDTTIQQGISGLIDSLNARDISVKIFEDFAQSWYWILVALGVAL 240CFPWTNVTPPALPGITNDTTIQQGISGLIDSLNARDISVKIFEDFAQSWYWILVA 24P4C12v.7: 181CFPWTNVTPPALPGITNDTTIQQGISGLIDSLNARDISVKIFEDFAQSWYWILVA----- 23524P4C12v.1: 241VLSLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISQLGFTTNLS 30024P4C12v.7: 235------------------------------------------------------------ 23524P4C12v.1: 301AYQSVQETWLAALIVLAVLEAILLLMLIFLRQRIRIAIALLKEASKAVGQMMSTMFYPLV 360                                               VGQMMSTMFYPLV 24P4C12v.7:236 -----------------------------------------------VGQMMSTMFYPLV 24824P4C12v.1: 361TFVLLLICIAYWAMTALYLATSGQPQYVLWASNISSPGCEKVPINTSCNPTAHLVNSSCP 420TFVLLLICIAYWAMTALYLATSGQPQYVLWASNISSPGCEKVPINTSCNPTAHLVNSSCP 24P4C12v.7:249 TFVLLLICIAYWAMTALYLATSGQPQYVLWASNISSPGCEKVPINTSCNPTAHLVNSSCP 30824P4C12v.1: 421GLMCVFQGYSSKGLIQRSVFNLQIYGVLGLFWTLNWVLALGQCVLAGAFASFYWAFHKPQ 480GLMCVFQGYSSKGLIQRSVFNLQIYGVLGLFWTLNWVLALGQCVLAGAFASFYWAFHKPQ 24P4C12v.7:309 GLMCVFQGYSSKGLIQRSVFNLQIYGVLGLFWTLNWVLALGQCVLAGAFASFYWAFHKPQ 36824P4C12v.1: 481DIPTFPLISAFIRTLRYHTGSLAFGALILTLVQIARVILEYIDHKLRGVQNPVARCIMCC 540DIPTFPLISAFIRTLRYHTGSLAFGALILTLVQIARVILEYIDHKLRGVQNPVARCIMCC 24P4C12v.7:369 DIPTFPLISAFIRTLRYHTGSLAFGALILTLVQIARVILEYIDHKLRGVQNPVARCIMCC 42824P4C12v.1: 541FKCCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSAKNAFMLLMRNIVRVVVLDKVTDLLLF 600FKCCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSAKNAFMLLMRNIVRVVVLDKVTDLLLF 24P4C12v.7:429 FKCCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSAKNAFMLLMRNIVRVVVLDKVTDLLLF 48824P4C12v.1: 601FGKLLVVGGVGVLSFFFFSGRIPGLGKDFKSPHLNYYWLPIMTSILGAYVIASGFFSVFG 660FGKLLVVGGVGVLSFFFFSGRIPGLGKDFKSPHLNYYWLPIMTSILGAYVIASGFFSVFG 24P4C12v.7:489 FGKLLVVGGVGVLSFFFFSGRIPGLGKDFKSPHLNYYWLPIMTSILGAYVIASGFFSVFG 54824P4C12v.1: 661 MCVDTLFLCFLEDLERNNGSLDRPYYMSKSLLKILGKKNEAPPDNKKRKK 710MCVDTLFLCFLEDLERNNGSLDRPYYMSKSLLKILGKKNEAPPDNKKRKK 24P4C12v.7: 549MCVDTLFLCFLEDLERNNGSLDRPYYMSKSLLKILGKKNEAPPDNKKRKK 598

TABLE LVI Nucleotide sequence of transcript variant 24P4C12 v.8 (SEQ IDNO: 100) gagccatggg gggaaagcag cgggacgagg atgacgaggc ctacgggaagccagtcaaat 60 acgacccctc ctttcgaggc cccatcaaga acagaagctg cacagatgtcatctgctgcg 120 tcctcttcct gctcttcatt ctaggttaca tcgtggtggg gattgtggcctggttgtatg 180 gagacccccg gcaagtcctc taccccagga actctactgg ggcctactgtggcatggggg 240 agaacaaaga taagccgtat ctcctgtact tcaacatctt cagctgcatcctgtccagca 300 acatcatctc agttgctgag aacggcctac agtgccccac accccaggtgtgtgtgtcct 360 cctgcccgga ggacccatgg actgtgggaa aaaacgagtt ctcacagactgttggggaag 420 tcttctatac aaaaaacagg aacttttgtc tgccaggggt accctggaatatgacggtga 480 tcacaagcct gcaacaggaa ctctgcccca gtttcctcct cccctctgctccagctctgg 540 ggcgctgctt tccatggacc aacgttactc caccggcgct cccagggatcaccaatgaca 600 ccaccataca gcaggggatc agcggtctta ttgacagcct caatgcccgagacatcagtg 660 ttaagatctt tgaagatttt gcccagtcct ggtattggat tcttgttgccctgggggtgg 720 ctctggtctt gagcctactg tttatcttgc ttctgcgcct ggtggctgggcccctggtgc 780 tggtgctgat cctgggagtg ctgggcgtgc tggcatacgg catctactactgctgggagg 840 agtaccgagt gctgcgggac aagggcgcct ccatctccca gctgggtttcaccaccaacc 900 tcagtgccta ccagagcgtg caggagacct ggctggccgc cctgatcgtgttggcggtgc 960 ttgaagccat cctgctgctg atgctcatct tcctgcggca gcggattcgtattgccatcg 1020 ccctcctgaa ggaggccagc aaggctgtgg gacagatgat gtctaccatgttctacccac 1080 tggtcacctt tgtcctcctc ctcatctgca ttgcctactg ggccatgactgctctgtacc 1140 tggctacatc ggggcaaccc cagtatgtgc tctgggcatc caacatcagctcccccggct 1200 gtgagaaagt gccaataaat acatcatgca accccacggc ccaccttgtgaactcctcgt 1260 gcccagggct gatgtgcgtc ttccagggct actcatccaa aggcctaatccaacgttctg 1320 tcttcaatct gcaaatctat ggggtcctgg ggctcttctg gacccttaactgggtactgg 1380 ccctgggcca atgcgtcctc gctggagcct ttgcctcctt ctactgggccttccacaagc 1440 cccaggacat ccctaccttc cccttaatct ctgccttcat ccgcacactccgttaccaca 1500 ctgggtcatt ggcatttgga gccctcatcc tgacccttgt gcagatagcccgggtcatct 1560 tggagtatat tgaccacaag ctcagaggag tgcagaaccc tgtagcccgctgcatcatgt 1620 gctgtttcaa gtgctgcctc tggtgtctgg aaaaatttat caagttcctaaaccgcaatg 1680 catacatcat gatcgccatc tacgggaaga atttctgtgt ctcagccaaaaatgcgttca 1740 tgctactcat gcgaaacatt gtcagggtgg tcgtcctgga caaagtcacagacctgctgc 1800 tgttctttgg gaagctgctg gtggtcggag gcgtgggggt cctgtccttcttttttttct 1860 ccggtcgcat cccggggctg ggtaaagact ttaagagccc ccacctcaactattactggc 1920 tgcccatcat gaggaaccca ataaccccaa cgggtcatgt cttccagacctccatcctgg 1980 gggcctatgt catcgccagc ggcttcttca gcgttttcgg catgtgtgtggacacgctct 2040 tcctctgctt cctggaagac ctggagcgga acaacggctc cctggaccggccctactaca 2100 tgtccaagag ccttctaaag attctgggca agaagaacga ggcgcccccggacaacaaga 2160 agaggaagaa gtgacagctc cggccctgat ccaggactgc accccacccccaccgtccag 2220 ccatccaacc tcacttcgcc ttacaggtct ccattttgtg gtaaaaaaaggttttaggcc 2280 aggcgccgtg gctcacgcct gtaatccaac actttgagag gctgaggcgggcggatcacc 2340 tgagtcagga gttcgagacc agcctggcca acatggtgaa acctccgtctctattaaaaa 2400 tacaaaaatt agccgagagt ggtggcatgc acctgtcatc ccagctactcgggaggctga 2460 ggcaggagaa tcgcttgaac ccgggaggca gaggttgcag tgagccgagatcgcgccact 2520 gcactccaac ctgggtgaca gactctgtct ccaaaacaaa acaaacaaacaaaaagattt 2580 tattaaagat attttgttaa ctcagtaaaa aaaaaaaaaa aaa 2623

TABLE LVIII Peptide sequences of protein coded by 24P4C12 v.8 (SEQ IDNO:103) MGGKQRDEDD EAYGKPVKYD PSFRGPIKNR SCTDVICCVL FLLFILGYIVVGIVAWLYGD 60 PRQVLYPRNS TGAYCGMGEN KDKPYLLYFN IFSCILSSNI ISVAENGLQCPTPQVCVSSC 120 PEDPWTVGKN EFSQTVGEVF YTKNRNFCLP GVPWNMTVIT SLQQELCPSFLLPSAPALGR 180 CFPWTNVTPP ALPGITNDTT IQQGISGLID SLNARDISVK IFEDFAQSWYWILVALGVAL 240 VLSLLFILLL RLVAGPLVLV LILGVLGVLA YGIYYCWEEY RVLRDKGASISQLGFTTNLS 300 AYQSVQETWL AALIVLAVLE AILLLMLIFL RQRIRIAIAL LKEASKAVGQMMSTMFYPLV 360 TFVLLLICIA YWAMTALYLA TSGQPQYVLW ASNISSPGCE KVPINTSCNPTAHLVNSSCP 420 GLMCVFQGYS SKGLIQRSVF NLQIYGVLGL FWTLNWVLAL GQCVLAGAFASFYWAFHKPQ 480 DIPTFPLISA FIRTLRYHTG SLAFGALILT LVQIARVILE YIDHKLRGVQNPVARCIMCC 540 FKCCLWCLEK FIKFLNRNAY IMIAIYGKNF CVSAKNAFML LMRNIVRVVVLDKVTDLLLF 600 FGKLLVVGGV GVLSFFFFSG RIPGLGKDFK SPHLNYYWLP IMRNPITPTGHVFQTSILGA 660 YVIASGFFSV FGMCVDTLFL CFLEDLERNN GSLDRPYYMS KSLLKILGKKNEAPPDNKKR 720 KK 722

TABLE LIX Amino acid sequence alignment of 24P4C12v.1 v.1 (SEQ ID NO:104) and 24P4C12 v.8 (SEQ ID NO: 105) Score = 1438 bits (3722), Expect= 0.0Identities = 710/722 (98%), Positives = 710/722 (98%), Gaps= 12/722 (1%) 24P4C12v.1: 1MGGKQRDEDDEAYGKPVKYDPSFRGPIKNRSCTDVICCVLFLLFILGYIVVGIVAWLYGD 60MGGKQRDEDDEAYGKPVKYDPSFRGPIKNRSCTDVICCVLFLLFILGYIVVGIVAWLYGD 24P4C12v.8:1 MGGKQRDEDDEAYGKPVKYDPSFRGPIKNRSCTDVICCVLFLLFILGYIVVGIVAWLYGD 6024P4C12v.1: 61PRQVLYPRNSTGAYCGMGENKDKPYLLYFNIFSCILSSNIISVAENGLQCPTPQVCVSSC 120PRQVLYPRNSTGAYCGMGENKDKPYLLYFNIFSCILSSNIISVAENGLQCPTPQVCVSSC 24P4C12v.8:61 PRQVLYPRNSTGAYCGMGENKDKPYLLYFNIFSCILSSNIISVAENGLQCPTPQVCVSSC 12024P4C12v.1: 121PEDPWTVGKNEFSQTVGEVFYTKNRNFCLPGVPWNMTVITSLQQELCPSFLLPSAPALGR 180PEDPWTVGKNEFSQTVGEVFYTKNRNFCLPGVPWNMTVITSLQQELCPSFLLPSAPALGR 24P4C12v.8:121 PEDPWTVGKNEFSQTVGEVFYTKNRNFCLPGVPWNMTVITSLQQELCPSFLLPSAPALGR 18024P4C12v.1: 181CFPWTNVTPPALPGITNDTTIQQGISGLIDSLNARDISVKIFEDFAQSWYWILVALGVAL 240CFPWTNVTPPALPGITNDTTIQQGISGLIDSLNARDISVKIFEDFAQSWYWILVALGVAL 24P4C12v.8:181 CFPWTNVTPPALPGITNDTTIQQGISGLIDSLNARDISVKIFEDFAQSWYWILVALGVAL 24024P4C12v.1: 241VLSLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISQLGFTTNLS 300VLSLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISQLGFTTNLS 24P4C12v.8:241 VLSLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISQLGFTTNLS 30024P4C12v.1: 301AYQSVQETWLAALIVLAVLEAILLLMLIFLRQRIRIAIALLKEASKAVGQMMSTMFYPLV 360AYQSVQETWLAALIVLAVLEAILLLMLIFLRQRIRIAIALLKEASKAVGQMMSTMFYPLV 24P4C12v.8:301 AYQSVQETWLAALIVLAVLEAILLLMLIFLRQRIRIAIALLKEASKAVGQMMSTMFYPLV 36024P4C12v.1: 361TFVLLLICIAYWAMTALYLATSGQPQYVLWASNISSPGCEKVPINTSCNPTAHLVNSSCP 420TFVLLLICIAYWAMTALYLATSGQPQYVLWASNISSPGCEKVPINTSCNPTAHLVNSSCP 24P4C12v.8:361 TFVLLLICIAYWAMTALYLATSGQPQYVLWASNISSPGCEKVPINTSCNPTAHLVNSSCP 42024P4C12v.1: 421GLMCVFQGYSSKGLIQRSVFNLQIYGVLGLFWTLNWVLALGQCVLAGAFASFYWAFHKPQ 480GLMCVFQGYSSKGLIQRSVFNLQIYGVLGLFWTLNWVLALGQCVLAGAFASFYWAFHKPQ 24P4C12v.8:421 GLMCVFQGYSSKGLIQRSVFNLQIYGVLGLFWTLNWVLALGQCVLAGAFASFYWAFHKPQ 48024P4C12v.1: 481DIPTFPLISAFIRTLRYHTGSLAFGALILTLVQIARVILEYIDHKLRGVQNPVARCIMCC 540DIPTFPLISAFIRTLRYHTGSLAFGALILTLVQIARVILEYIDHKLRGVQNPVARCIMCC 24P4C12v.8:481 DIPTFPLISAFIRTLRYHTGSLAFGALILTLVQIARVILEYIDHKLRGVQNPVARCIMCC 54024P4C12v.1: 541FKCCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSAKNAFMLLMRNIVRVVVLDKVTDLLLF 600FKCCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSAKNAFMLLMRNIVRVVVLDKVTDLLLF 24P4C12v.8:541 FKCCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSAKNAFMLLMRNIVRVVVLDKVTDLLLF 60024P4C12v.1: 601FGKLLVVGGVGVLSFFFFSGRIPGLGKDFKSPHLNYYWLPIM------------TSILGA 648FGKLLVVGGVGVLSFFFFSGRIPGLGKDFKSPHLNYYWLPIM            TSILGA 24P4C12v.8:601 FGKLLVVGGVGVLSFFFFSGRIPGLGKDFKSPHLNYYWLPIMRNPITPTGHVFQTSILGA 66024P4C12v1: 649YVIASGFFSVFGMCVDTLFLCFLEDLERNNGSLDRPYYMSKSLLKILGKKNEAPPDNKKR 708YVIASGFFSVFGMCVDTLFLCFLEDLERNNGSLDRPYYMSKSLLKILGKKNEAPPDNKKR 24P4C12v.8:661 YVIASGFFSVFGMCVDTLFLCFLEDLERNNGSLDRPYYMSKSLLKILGKKNEAPPDNKKR 72024P4C12v.1: 709 KK 710 KK 24P4C12v.8: 721 KK 722

TABLE LX Nucleotide sequence of transcript variant 24P4C12 v.9 (SEQ IDNO:106) gagccatggg gggaaagcag cgggacgagg atgacgaggc ctacgggaagccagtcaaat 60 acgacccctc ctttcgaggc cccatcaaga acagaagctg cacagatgtcatctgctgcg 120 tcctcttcct gctcttcatt ctaggttaca tcgtggtggg gattgtggcctggttgtatg 180 gagacccccg gcaagtcctc taccccagga actctactgg ggcctactgtggcatggggg 240 agaacaaaga taagccgtat ctcctgtact tcaacatctt cagctgcatcctgtccagca 300 acatcatctc agttgctgag aacggcctac agtgccccac accccaggtgtgtgtgtcct 360 cctgcccgga ggacccatgg actgtgggaa aaaacgagtt ctcacagactgttggggaag 420 tcttctatac aaaaaacagg aacttttgtc tgccaggggt accctggaatatgacggtga 480 tcacaagcct gcaacaggaa ctctgcccca gtttcctcct cccctctgctccagctctgg 540 ggcgctgctt tccatggacc aacgttactc caccggcgct cccagggatcaccaatgaca 600 ccaccataca gcaggggatc agcggtctta ttgacagcct caatgcccgagacatcagtg 660 ttaagatctt tgaagatttt gcccagtcct ggtattggat tcttgttgccctgggggtgg 720 ctctggtctt gagcctactg tttatcttgc ttctgcgcct ggtggctgggcccctggtgc 780 tggtgctgat cctgggagtg ctgggcgtgc tggcatacgg catctactactgctgggagg 840 agtaccgagt gctgcgggac aagggcgcct ccatctccca gctgggtttcaccaccaacc 900 tcagtgccta ccagagcgtg caggagacct ggctggccgc cctgatcgtgttggcggtgc 960 ttgaagccat cctgctgctg atgctcatct tcctgcggca gcggattcgtattgccatcg 1020 ccctcctgaa ggaggccagc aaggctgtgg gacagatgat gtctaccatgttctacccac 1080 tggtcacctt tgtcctcctc ctcatctgca ttgcctactg ggccatgactgctctgtatc 1140 ctctgcccac gcagccagcc actcttggat atgtgctctg ggcatccaacatcagctccc 1200 ccggctgtga gaaagtgcca ataaatacat catgcaaccc cacggcccaccttgtgaact 1260 cctcgtgccc agggctgatg tgcgtcttcc agggctactc atccaaaggcctaatccaac 1320 gttctgtctt caatctgcaa atctatgggg tcctggggct cttctggacccttaactggg 1380 tactggccct gggccaatgc gtcctcgctg gagcctttgc ctccttctactgggccttcc 1440 acaagcccca ggacatccct accttcccct taatctccgc cttcatccgcacactccgtt 1500 accacactgg gtcattggca tttggagccc tcatcctgac ccttgtgcagatagcccggg 1560 tcatcttgga gtatattgac cacaagctca gaggagtgca gaaccctgtagcccgctgca 1620 tcatgtgctg tttcaagtgc tgcctctggt gtctggaaaa atttatcaagttcctaaacc 1680 gcaatgcata catcatgatc gccatctacg ggaagaattt ctgtgtctcagccaaaaatg 1740 cgttcatgct actcatgcga aacattgtca gggtggtcgt cctggacaaagtcacagacc 1800 tgctgctgtt ctttgggaag ctgctggtgg tcggaggcgt gggggtcctgtccttctttt 1860 ttttctccgg tcgcatcccg gggctgggta aagactttaa gagcccccacctcaactatt 1920 actggctgcc catcatgacc tccatcctgg gggcctatgt catcgccagcggcttcttca 1980 gcgttttcgg catgtgtgtg gacacgctct tcctctgctt cctggaagacctggagcgga 2040 acaacggctc cctggaccgg ccctactaca tgtccaagag ccttctaaagattctgggca 2100 agaagaacga ggcgcccccg gacaacaaga agaggaagaa gtgacagctccggccctgat 2160 ccaggactgc accccacccc caccgtccag ccatccaacc tcacttcgccttacaggtct 2220 ccattttgtg gtaaaaaaag gttttaggcc aggcgccgtg gctcacgcctgtaatccaac 2280 actttgagag gctgaggcgg gcggatcacc tgagtcagga gttcgagaccagcctggcca 2340 acatggtgaa acctccgtct ctattaaaaa tacaaaaatt agccgagagtggtggcatgc 2400 acctgtcatc ccagctactc gggaggctga ggcaggagaa tcgcttgaacccgggaggca 2460 gaggttgcag tgagccgaga tcgcgccact gcactccaac ctgggtgacagactctgtct 2520 ccaaaacaaa acaaacaaac aaaaagattt tattaaagat attttgttaactcagtaaaa 2580 aaaaaaaaaa aaa 2593

TABLE LXII Peptide sequences of protein coded by 24P4C12 v.9 (SEQ ID NO:109) MGGKQRDEDD EAYGKPVKYD PSFRGPIKNR SCTDVICCVL FLLFILGYIV VGIVAWLYGD60 PRQVLYPRNS TGAYCGMGEN KDKPYLLYFN IFSCILSSNI ISVAENGLQC PTPQVCVSSC 120PEDPWTVGKN EFSQTVGEVF YTKNRNFCLP GVPWNMTVIT SLQQELCPSF LLPSAPALGR 180CFPWTNVTPP ALPGITNDTT IQQGISGLID SLNARDISVK IFEDFAQSWY WILVALGVAL 240VLSLLFILLL RLVAGPLVLV LILGVLGVLA YGIYYCWEEY RVLRDKGASI SQLGFTTNLS 300AYQSVQETWL AALIVLAVLE AILLLMLIFL RQRIRIAIAL LKEASKAVGQ MMSTMFYPLV 360TFVLLLICIA YWAMTALYPL PTQPATLGYV LWASNISSPG CEKVPINTSC NPTAHLVNSS 420CPGLMCVFQG YSSKGLIQRS VFNLQIYGVL GLFWTLNWVL ALGQCVLAGA FASFYWAFHK 480PQDIPTFPLI SAFIRTLRYH TGSLAFGALI LTLVQIARVI LEYIDHKLRG VQNPVARCIM 540CCFKCCLWCL EKFIKFLNRN AYIMIAIYGK NFCVSAKNAF MLLMRNIVRV VVLDKVTDLL 600LFFGKLLVVG GVGVLSFFFF SGRIPGLGKD FKSPHLNYYW LPIMTSILGA YVIASGFFSV 660FGMCVDTLFL CFLEDLERNN GSLDRPYYMS KSLLKILGKK NEAPPDNKKR KK 712

TABLE LXIII Amino acid sequence alignment of 24P4C12v.1 v.1 (SEQ ID NO:110) and 24P4C12 v.9 (SEQ ID NO: 111) Score = 1424 bits (3686), Expect= 0.0Identities = 704/713 (98%), Positives = 705/713 (98%), Gaps = 4/713(0%) 24P4C12v.1: 1MGGKQRDEDDEAYGKPVKYDPSFRGPIKNRSCTDVICCVLFLLFILGYIVVGIVAWLYGD 60MGGKQRDEDDEAYGKPVKYDPSFRGPIKNRSCTDVICCVLFLLFILGYIVVGIVAWLYGD 24P4C12v.9:1 MGGKQRDEDDEAYGKPVKYDPSFRGPIKNRSCTDVICCVLFLLFILGYIVVGIVAWLYGD 6024P4C12v.1: 61PRQVLYPRNSTGAYCGMGENKDKPYLLYFNIFSCILSSNIISVAENGLQCPTPQVCVSSC 120PRQVLYPRNSTGAYCGMGENKDKPYLLYFNIFSCILSSNIISVAENGLQCPTPQVCVSSC 24P4C12v.9:61 PRQVLYPRNSTGAYCGMGENKDKPYLLYFNIFSCILSSNIISVAENGLQCPTPQVCVSSC 12024P4C12v.1: 121PEDPWTVGKNEFSQTVGEVFYTKNRNFCLPGVPWNMTVITSLQQELCPSFLLPSAPALGR 180PEDPWTVGKNEFSQTVGEVFYTKNRNFCLPGVPWNMTVITSLQQELCPSFLLPSAPALGR 24P4C12v.9:121 PEDPWTVGKNEFSQTVGEVFYTKNRNFCLPGVPWNMTVITSLQQELCPSFLLPSAPALGR 18024P4C12v.1: 181CFPWTNVTPPALPGITNDTTIQQGISGLIDSLNARDISVKIFEDFAQSWYWILVALGVAL 240CFPWTNVTPPALPGITNDTTIQQGISGLIDSLNARDISVKIFEDFAQSWYWILVALGVAL 24P4C12v.9:181 CFPWTNVTPPALPGITNDTTIQQGISGLIDSLNARDISVKIFEDFAQSWYWILVALGVAL 24024P4C12v.1: 241VLSLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISQLGFTTNLS 300VLSLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISQLGFTTNLS 24P4C12v.9:241 VLSLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISQLGFTTNLS 30024P4C12v.1: 301AYQSVQETWLAALIVLAVLEAILLLMLIFLRQRIRIAIALLKEASKAVGQMMSTMFYPLV 360AYQSVQETWLAALIVLAVLEAILLLMLIFLRQRIRIAIALLKEASKAVGQMMSTMFYPLV 24P4C12v.9:301 AYQSVQETWLAALIVLAVLEAILLLMLIFLRQRIRIAIALLKEASKAVGQMMSTMFYPLV 36024P4C12v.1: 361TFVLLLICIAYWAMTALYLATSGQPQ---YVLWASNISSPGCEKVPINTSCNPTAHLVNS 417TFVLLLICIAYWAMTALY   + QP    YVLWASNISSPGCEKVPINTSCNPTAHLVNS 24P4C12v.9:361 TFVLLLICIAYWAMTALYPLPT-QPATLGYVLWASNISSPGCEKVPINTSCNPTAHLVNS 41924P4C12v.1: 418SCPGLMCVFQGYSSKGLIQRSVFNLQIYGVLGLFWTLNWVLALGQCVLAGAFASFYWAFH 477SCPGLMCVFQGYSSKGLIQRSVFNLQIYGVLGLFWTLNWVLALGQCVLAGAFASFYWAFH 24P4C12v.9:420 SCPGLMCVFQGYSSKGLIQRSVFNLQIYGVLGLFWTLNWVLALGQCVLAGAFASFYWAFH 47924P4C12v.1: 478KPQDIPTFPLISAFIRTLRYHTGSLAFGALILTLVQIARVILEYIDHKLRGVQNPVARCI 537KPQDIPTFPLISAFIRTLRYHTGSLAFGALILTLVQIARVILEYIDHKLRGVQNPVARCI 24P4C12v.9:480 KPQDIPTFPLISAFIRTLRYHTGSLAFGALILTLVQIARVILEYIDHKLRGVQNPVARCI 53924P4C12v.1: 538MCCFKCCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSAKNAFMLLMRNIVRVVVLDKVTDL 597MCCFKCCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSAKNAFMLLMRNIVRVVVLDKVTDL 24P4C12v.9:540 MCCFKCCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSAKNAFMLLMRNIVRVVVLDKVTDL 59924P4C12v.1: 598LLFFGKLLVVGGVGVLSFFFFSGRIPGLGKDFKSPHLNYYWLPIMTSILGAYVIASGFFS 657LLFFGKLLVVGGVGVLSFFFFSGRIPGLGKDFKSPHLNYYWLPIMTSILGAYVIASGFFS 24P4C12v.9:600 LLFFGKLLVVGGVGVLSFFFFSGRIPGLGKDFKSPHLNYYWLPIMTSILGAYVIASGFFS 65924P4C12v.1: 658 VFGMCVDTLFLCFLEDLERNNGSLDRPYYMSKSLLKILGKKNEAPPDNKKRKK710 VFGMCVDTLFLCFLEDLERNNGSLDRPYYMSKSLLKILGKKNEAPPDNKKRKK 24P4C12v.9:660 VFGMCVDTLFLCFLEDLERNNGSLDRPYYMSKSLLKILGKKNEAPPDNKKRKK 712

1-22. (canceled)
 23. Antibodies or antigen binding fragments thereofspecifically immunoreactive with a 24P4C12 protein that has the aminoacid sequence of SEQ ID NO: 3 or is an allelic variant thereof that hasan amino acid sequence at least 90% identical to SEQ ID NO: 3 or has theamino acid sequence shown in SEQ ID NO:
 15. 24. The antibodies orfragments of claim 23, wherein the 24P4C12 protein has the amino acidsequence shown in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ IDNO:
 19. 25. The antibodies or fragments of claim 23 which arepolyclonal.
 26. The antibodies or fragments of claim 23 which aremonoclonal.
 27. The fragments of claim 23 which are Fab, F(ab)₂, orF_(v) fragments.
 28. The antibodies or fragments of claim 26 which aresingle chain antibodies.
 29. The antibodies or fragments of claim 26which are composed of portions of human and murine origin.
 30. Theantibodies or fragments of claim 23 which comprise human CDR's.
 31. Theantibodies or fragments of claim 23 which are human or humanized. 32.The antibodies or fragments of claim 23 which are labeled with adetectable marker.
 33. The antibodies or fragments of claim 32 whereinthe detectable marker is a radio isotope, a fluorescent compound, abioluminescent compound, a chemiluminescent compound, a metal chelatoror an enzyme.
 34. The antibodies or fragments of claim 23 that arecoupled to a toxin, therapeutic agent or a solid matrix.
 35. Theantibodies or fragments of claim 23 that are coupled to a toxin. 36.Hybridomas that produce the monoclonal antibodies of claim
 26. 37.Recombinant host cells that produce the monoclonal antibodies orfragments of claim
 26. 38. Recombinant host cells that produce thesingle chain antibodies of claim
 28. 39. A method to purify apolypeptide that binds to the antibodies or fragments of claim 23 whichmethod comprises contacting a sample containing said polypeptide withthe antibodies or fragments so as to form a complex between saidantibodies or fragments and said polypeptide; and separating theresultant complex from the sample.